Aberrant mitochondrial dna, associated fusion transcripts and hybridization probes therefor

ABSTRACT

The present invention provides novel mitochondrial fusion transcripts and the parent mutated mtDNA molecules that are useful for predicting, diagnosing and/or monitoring cancer. Hybridization probes complementary thereto for use in the methods of the invention are also provided.

FIELD OF THE INVENTION

The present invention relates to the field of mitochondrial genomics. Inone aspect, the invention relates to the identification and use ofmitochondrial genome fusion transcripts and probes that hybridizethereto.

BACKGROUND OF THE INVENTION

Mitochondrial Genome

The mitochondrial genome is a compact yet critical sequence of nucleicacids. Mitochondrial DNA, or “mtDNA”, comprises a small genome of 16,569nucleic acid base pairs (bp) (Anderson et al., 1981; Andrews et al.,1999) in contrast to the immense nuclear genome of 3.3 billion by(haploid). Its genetic complement is substantially smaller than that ofits nuclear cell mate (0.0005%). However, individual cells carryanywhere from 10³ to 10⁴ mitochondria depending on specific cellularfunctions (Singh and Modica-Napolitano 2002). Communication or chemicalsignalling routinely occurs between the nuclear and mitochondrialgenomes (Sherratt et al., 1997). Moreover, specific nuclear componentsare responsible for the maintenance and integrity of mitochondrialsequences (Croteau et al., 1999). All mtDNA genomes in a givenindividual are identical due to the clonal expansion of mitochondriawithin the ovum, once fertilization has occurred. However mutagenicevents can induce sequence diversity reflected as somatic mutations.These mutations may accumulate in different tissues throughout the bodyin a condition known as heteroplasmy.

Mitochondrial Proteome

About 3,000 nuclear genes are required to construct, operate andmaintain mitochondria, with only thirty-seven of these coded by themitochondrial genome, indicating heavy mitochondrial dependence onnuclear loci. The mitochondrial genome codes for a complement of 24genes, including 2 rRNAs and 22 tRNAs that ensure correct translation ofthe remaining 13 genes which are vital to electron transport (see FIG.1). The mitochondrial genome is dependent on seventy nuclear encodedproteins to accomplish the oxidation and reduction reactions necessaryfor this vital function, in addition to the thirteen polypeptidessupplied by the mitochondrial genome. Both nuclear and mitochondrialproteins form complexes spanning the inner mitochondrial membrane andcollectively generate 80-90% of the chemical fuel adenosinetriphosphate, or ATP, required for cellular metabolism. In addition toenergy production, mitochondria play a central role in other metabolicpathways as well. A critical function of the mitochondria is mediationof cell death, or apoptosis (see Green and Kroemer, 2005). Essentially,there are signal pathways which permeabilize the outer mitochondrialmembrane, or in addition, the inner mitochondrial membrane as well. Whenparticular mitochondrial proteins are released into the cytosol,non-reversible cell death is set in motion. This process highlights themulti-functional role that some mitochondrial proteins have. Thesemulti-tasking proteins suggest that there are other mitochondrialproteins as well which may have alternate functions.

Mitochondrial Fusion Transcriptome

The mitochondrial genome is unusual in that it is a circular,intron-less DNA molecule. The genome is interspersed with repeat motifswhich flank specific lengths of sequences. Sequences between theserepeats are prone to deletion under circumstances which are not wellunderstood. Given the number of repeats in the mitochondrial genome,there are many possible deletions. The best known example is the 4977“common deletion.” This deletion has been associated with severalpurported conditions and diseases and is thought to increase infrequency with aging (Dai et al., 2004; Ro et al., 2003; Barron et al.,2001; Lewis et al., 2000; Muller-Hocker, 1998; Porteous et al., 1998)(FIG. 4). The current thinking in the field of mitochondrial genomics isthat mitochondrial deletions are merely deleterious by-products ofdamage to the mitochondrial genome by such agents as reactive oxygenspecies and UVR. (Krishnan et al 2008, Nature Genetics). Further, thoughit is recognized that high levels of mtDNA deletions can have severeconsequences on the cell's ability to produce energy in the form of ATPas a result of missing gene sequences necessary for cellularrespiration, it is not anticipated that these deleted mitochondrialmolecules may be a component of downstream pathways, have an intendedfunctional role, and possibly may be more aptly viewed as alternatenatural forms of the recognized genes of the mitochondria as has beenanticipated by the Applicant.

The sequence dynamics of mtDNA are important diagnostic tools. Mutationsin mtDNA are often preliminary indicators of developing disease. Forexample, it has been demonstrated that point mutations in themitochondrial genome are characteristic of tumour foci in the prostate.This trend also extends to normal appearing tissue both adjacent to anddistant from tumour tissue (Parr et al. 2006). This suggests thatmitochondrial mutations occur early in the malignant transformationpathway.

For example, the frequency of a 3.4 kb mitochondrial deletion hasexcellent utility in discriminating between benign and malignantprostate tissues (Maki et al. 2008).

Mitochondrial fusion transcripts have been reported previously in theliterature, first in soybeans (Morgens et al. 1984) and then later intwo patients with Kearns-Sayre Syndrome, a rare neuromuscular disorder(Nakase et al 1990). Importantly, these transcripts were not found tohave (or investigated regarding) association with any human cancers.

SUMMARY OF THE INVENTION

An object of the present invention to provide aberrant mitochondrialDNA, associated fusion transcripts and hybridization probes therefor.

In accordance with an aspect of the invention, there is provided anisolated mitochondrial fusion transcript associated with cancer.

In accordance with an aspect of the invention, there is provided amitochondrial fusion protein corresponding to the above fusiontranscript, having a sequence as set forth in any one of SEQ ID NOs: 34to 49 and 52.

In accordance with another aspect of the invention, there is provided anisolated mtDNA encoding a fusion transcript of the invention.

In accordance with another aspect of the invention, there is provided ahybridization probe having a nucleic acid sequence complementary to atleast a portion of a mitochondrial fusion transcript or an mtDNA of theinvention.

In accordance with another aspect of the invention, there is provided amethod of detecting a cancer in a mammal, the method comprising assayinga tissue sample from the mammal for the presence of at least onemitochondrial fusion transcript associated with cancer by hybridizingthe sample with at least one hybridization probe having a nucleic acidsequence complementary to at least a portion of a mitochondrial fusiontranscript according to the invention.

In accordance with another aspect of the invention, there is provided amethod of detecting a cancer in a mammal, the method comprising assayinga tissue sample from the mammal for the presence of at least oneaberrant mtDNA associated with cancer by hybridizing the sample with atleast one hybridization probe having a nucleic acid sequencecomplementary to at least a portion of an mtDNA according to theinvention.

In accordance with another aspect of the invention, there is provided akit for conducting an assay for detecting the presence of a cancer in amammal, said kit comprising at least one hybridization probecomplementary to at least a portion of a fusion transcript or an mtDNAof the invention.

In accordance with another aspect of the invention, there is provided ascreening tool comprised of a microarray having 10's, 100's, or 1000'sof mitochondrial fusion transcripts for identification of thoseassociated with cancer.

In accordance with another aspect of the invention, there is provided ascreening tool comprised of a microarray having 10's, 100's, or 1000'sof mitochondrial DNAs corresponding to mitochondrial fusion transcriptsfor identification of those associated with cancer.

In accordance with another aspect of the invention, there is provided ascreening tool comprised of a multiplexed branched DNA assay having10's, 100's, or 1000's of mitochondrial fusion transcripts foridentification of those associated with cancer.

In accordance with another aspect of the invention, there is provided ascreening tool comprised of a multiplexed branched DNA assay having10's, 100's, or 1000's of mitochondrial DNAs corresponding tomitochondrial fusion transcripts for identification of those associatedwith cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the invention will now be described by way of exampleonly with reference to the appended drawings wherein:

FIG. 1 is an illustration showing mitochondrial coding genes.

FIG. 2 shows polyadenalated fusion transcripts in prostate samplesinvoked by the loss of the 3.4 kb deletion.

FIG. 3 shows polyadenalated fusion transcripts in prostate samplesinvoked by the loss of the 4977 kb common deletion.

FIG. 4 shows polyadenalated fusion transcripts in breast samples invokedby the loss of the 3.4 kb segment from the mtgenome.

FIGS. 5 a and 5 b show an example of a mitochondrial DNA region beforeand after splicing of genes.

FIGS. 6 a to 6 g illustrate the results for transcripts 2, 3, 8, 9, 10,11 and 12 of the invention in the identification of colorectal cancertumours.

FIGS. 7 a to 7 d illustrate the results for transcripts 6, 8, 10 and 20of the invention in the identification of lung cancer tumours.

FIGS. 8 a to 8 g illustrate the results for transcripts 6, 10, 11, 14,15, 16 and 20 of the invention in the identification of melanomas.

FIGS. 9 a to 9 h illustrate the results for transcripts 1, 2, 3, 6, 11,12, 15 and 20 of the invention in the identification of ovarian cancer.

FIGS. 10 to 18 illustrate the results for transcripts 2, 3, 4, 11, 12,13, 15, 16 and 20 of the invention in the identification of testicularcancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel mitochondrial fusion transcriptsand the parent mutated mtDNA molecules that are useful for predicting,diagnosing and/or monitoring cancer. The invention further provideshybridization probes for the detection of fusion transcripts andassociated mtDNA molecules and the use of such probes.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, “aberration” or “mutation” encompasses any modificationin the wild type mitochondrial DNA sequence that results in a fusiontranscript and includes, without limitation, insertions, translocations,deletions, duplications, recombinations, rearrangements or combinationsthereof.

As defined herein, “biological sample” refers to a tissue or bodilyfluid containing cells from which a molecule of interest can beobtained. For example, the biological sample can be derived from tissuesuch as prostate, breast, colorectal, lung and skin, or from blood,saliva, cerebral spinal fluid, sputa, urine, mucous, synovial fluid,peritoneal fluid, amniotic fluid and the like. The biological sample maybe a surgical specimen or a biopsy specimen. The biological sample canbe used either directly as obtained from the source or following apre-treatment to modify the character of the sample. Thus, thebiological sample can be pre-treated prior to use by, for example,preparing plasma or serum from blood, disrupting cells, preparingliquids from solid materials, diluting viscous fluids, filteringliquids, distilling liquids, concentrating liquids, inactivatinginterfering components, adding reagents, and the like.

A “continuous” transcript is a fusion transcript that keeps the readingframe from the beginning to the end of both spliced genes. An “end”transcript is a fusion transcript that results in a prematuretermination codon before the original termination codon of a secondspliced gene.

As used herein, “mitochondrial DNA” or “mtDNA” is DNA present inmitochondria.

As used herein, the expression “mitochondrial fusion transcript” or“fusion transcript” refers to an RNA transcription product produced as aresult of the transcription of a mutated mitochondrial DNA sequencewherein such mutations may comprise mitochondrial deletions and otherlarge-scale mitochondrial DNA rearrangements.

Computer Analysis and Sequence Targetting

As discussed above, mitochondrial fusion transcripts have been reportedin soybeans (Morgens et al. 1984) and in humans suffering from a rareneuromuscular disorder (Nakase et al 1990). Fusion transcriptsassociated with human cancer have not, however, been described.

Using the knowledge gained from mapping the large-scale deletions of thehuman mitochondrial genome associated with cancer, the observation ofhigh frequencies of these deletions, and the evidence in anotherorganism and another disease type of trancriptionally active mutatedmtDNA molecules, Applicant hypothesized that such deletions may haveimportance beyond the DNA molecule and the damage and repair processesas it relates to cancer. To test this hypothesis computer analysis ofthe mitochondrial genome was conducted, specific for repeat elements,which suggested many potential deletion sites. Following this initialstep identifying unique repeats in the mitochondrial sequence havingnon-adjacent or non-tandem locations, a filter was then applied toidentify those repeats that upon initiating a deletion event in the DNAmolecule would then likely reclose or religate to produce a fused DNAsequence having an open reading frame (ORF). A subset of 18 moleculeswere then selected for targetting to investigate whether: 1) theyexisted in the natural biological state of humans and 2) they hadrelevance to malignancy. Results from these investigations are describedhereinafter.

Genomic Mutations

Mitochondrial DNA (mtDNA) dynamics are an important diagnostic tool.Mutations in mtDNA are often preliminary indicators of developingdisease and behave as biomarkers indicative of risk factors associatedwith disease onset. According to the present invention, large-scalerearrangement mutations in the mitochondrial genome result in thegeneration of fusion transcripts associated with cancer. Thus, the useof mtDNA encoding such transcripts and probes directed thereto for thedetection, diagnosis and monitoring of cancer is provided.

One of skill in the art will appreciate that the mtDNA molecules for usein the methods of the present invention may be derived through theisolation of naturally-occurring mutants or may be based on thecomplementary sequence of any of the fusion transcripts describedherein. Exemplary mtDNA sequences and fusion transcripts are disclosedin Applicant's U.S. priority application No. 61/040,616, hereinincorporated in its entirety by reference.

Detection of Mutant Genomic Sequences

Mutant mtDNA sequences according to the present invention may compriseany modification that results in the generation of a fusion transcript.Non-limiting examples of such modifications include insertions,translocations, deletions, duplications, recombinations, rearrangementsor combinations thereof. While the modification or change can varygreatly in size from only a few bases to several kilobases, preferablythe modification results in a substantive deletion or other large-scalegenomic aberration.

Extraction of DNA to detect the presence of such mutations may takeplace using art-recognized methods, followed by amplification of all ora region of the mitochondrial genome, and may include sequencing of themitochondrial genome, as described in Current Protocols in MolecularBiology. Alternatively, crude tissue homogenates may be used as well astechniques not requiring amplification of specific fragments ofinterest.

The step of detecting the mutations can be selected from any techniqueas is known to those skilled in the art. For example, analyzing mtDNAcan comprise selection of targets by branching DNA, sequencing themtDNA, amplifying mtDNA by PCR, Southern, Northern, WesternSouth-Western blot hybridizations, denaturing HPLC, hybridization tomicroarrays, biochips or gene chips, molecular marker analysis,biosensors, melting temperature profiling or a combination of any of theabove.

Any suitable means to sequence mitochondrial DNA may be used.Preferably, mtDNA is amplified by PCR prior to sequencing. The method ofPCR is well known in the art and may be performed as described in Mullisand Faloona, 1987, Methods Enzymol., 155: 335. PCR products can besequenced directly or cloned into a vector which is then placed into abacterial host. Examples of DNA sequencing methods are found in Brumley,R. L. Jr. and Smith, L. M., 1991, Rapid DNA sequencing by horizontalultrathin gel electrophoresis, Nucleic Acids Res. 19:4121-4126 andLuckey, J. A., et al, 1993, High speed DNA sequencing by capillary gelelectrophoresis, Methods Enzymol. 218: 154-172. The combined use of PCRand sequencing of mtDNA is described in Hopgood, R., et al, 1992,Strategies for automated sequencing of human mtDNA directly from PCRproducts, Biotechniques 13:82-92 and Tanaka, M. et al, 1996, Automatedsequencing of mtDNA, Methods Enzymol. 264:407-421.

Methods of selecting appropriate sequences for preparing various primersare also known in the art. For example, the primer can be prepared usingconventional solid-phase synthesis using commercially availableequipment, such as that available from Applied Biosystems USA Inc.(Foster City, Calif.), DuPont, (Wilmington, Del.), or Milligen (Bedford,Mass.).

According to an aspect of the invention, to determine candidate genomicsequences, a junction point of a sequence deletion is first identified.Sequence deletions are primarily identified by direct and indirectrepetitive elements which flank the sequence to be deleted at the 5′ and3′ end. The removal of a section of the nucleotides from the genomefollowed by the ligation of the genome results in the creation of anovel junction point.

Upon identification of the junction point, the nucleotides of the genesflanking the junction point are determined in order to identify aspliced gene. Typically the spliced gene comprises the initiation codonfrom the first gene and the termination codon of the second gene, andmay be expressed as a continuous transcript, i.e. one that keeps thereading frame from the beginning to the end of both spliced genes. It isalso possible that alternate initiation or termination codons containedwithin the gene sequences may be used as is evidenced by SEQ ID No:2 andSEQ ID No: 17 disclosed herein. Some known mitochondrial deletionsdiscovered to have an open reading frame (ORF) when the rearrangedsequences are rejoined at the splice site are provided in Table 1.

Exemplary mtDNA molecules for use in the methods of the presentinvention, which have been verified to exist in the lab, are providedbelow. These mtDNAs are based on modifications of the knownmitochondrial genome (SEQ ID NO: 1) and have been assigned a fusion or“FUS” designation, wherein A:B represents the junction point between thelast mitochondrial nucleotide of the first spliced gene and the firstmitochondrial nucleotide of the second spliced gene. The identificationof the spliced genes is provided in parentheses followed by thecorresponding sequence identifier. Where provided below, (AltMet) and(OrigMet) refer to alternate and original translation start sites,respectively.

-   -   FUS 8469:13447 (AltMet) (ATP synthase F0 subunit 8 to NADH        dehydrogenase subunit) (SEQ ID No: 2)    -   FUS 10744:14124 (NADH dehydrogenase subunit 4L (ND4L) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 3)    -   FUS 7974:15496 (Cytochrome c oxidase subunit II (COII) to        Cytochrome b (Cytb)) (SEQ ID No: 4)    -   FUS 7992:15730 (Cytochrome c oxidase subunit II (COII) to        Cytochrome b (Cytb)) (SEQ ID No: 5)    -   FUS 8210:15339 (Cytochrome c oxidase subunit II (COII) to        Cytochrome b (Cytb)) (SEQ ID No: 6)    -   FUS 8828:14896 (ATP synthase F0 subunit 6 (ATPase6) to        Cytochrome b (Cytb)) (SEQ ID No: 7)    -   FUS 10665:14856 (NADH dehydrogenase subunit 4L (ND4L) to        Cytochrome b (Cytb)) (SEQ ID No: 8)    -   FUS 6075:13799 (Cytochrome c oxidase subunit I (COI) to NADH de        hydrogenase subunit 5 (ND5)) (SEQ ID No: 9)    -   FUS 6325:13989 (Cytochrome c oxidase subunit I (COI) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 10)    -   FUS 7438:13476 (Cytochrome c oxidase subunit I (COI) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 11)    -   FUS 7775:13532 (Cytochrome c oxidase subunit II (COII) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 12)    -   FUS 8213:13991 (Cytochrome c oxidase subunit II (COII) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 13)    -   FUS 9191:12909 (ATP synthase F0 subunit 6 (ATPase6) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 14)    -   FUS 9574:12972 (Cytochrome c oxidase subunit III (COII) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 15)    -   FUS 10367:12829 (NADH dehydrogenase subunit 3 (ND3) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 16)    -   FUS 8469:13447 (OrigMet) (ATP synthase F0 subunit 8 to NADH        dehydrogenase subunit) (SEQ ID No: 17)    -   FUS 9144:13816 ((ATP synthase F0 subunit 6 (ATPase6) to NADH        dehydrogenase subunit 5 (ND5)) (SEQ ID No: 51)

The present invention also provides the use of variants or fragments ofthese sequences for predicting, diagnosing and/or monitoring cancer.

“Variant”, as used herein, refers to a nucleic acid differing from amtDNA sequence of the present invention, but retaining essentialproperties thereof. Generally, variants are overall closely similar,and, in many regions, identical to a select mtDNA sequence.Specifically, the variants of the present invention comprise at leastone of the nucleotides of the junction point of the spliced genes, andmay further comprise one or more nucleotides adjacent thereto. In oneembodiment of the invention, the variant sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to any one of the mtDNAsequences of the invention, or the complementary strand thereto.

In the present invention, “fragment” refers to a short nucleic acidsequence which is a portion of that contained in the disclosed genomicsequences, or the complementary strand thereto. This portion includes atleast one of the nucleotides comprising the junction point of thespliced genes, and may further comprise one or more nucleotides adjacentthereto. The fragments of the invention are preferably at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt, atleast about 50 nt, at least about 75 nt, or at least about 150 nt inlength. A fragment “at least 20 nt in length,” for example, is intendedto include 20 or more contiguous bases of any one of the mtDNA sequenceslisted above. In this context “about” includes the particularly recitedvalue, a value larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini. These fragments haveuses that include, but are not limited to, as diagnostic probes andprimers as discussed herein. Of course, larger fragments (e.g., 50, 150,500, 600, 2000 nucleotides) are also contemplated.

Thus, in specific embodiments of the invention, the mtDNA sequences areselected from the group consisting of:

SEQ ID NO: 2 (FUS 8469:13447; AltMet)

SEQ ID NO: 3 (FUS 10744:14124)

SEQ ID NO: 4 (FUS 7974:15496)

SEQ ID NO: 5 (FUS 7992:15730)

SEQ ID NO: 6 (FUS 8210:15339)

SEQ ID NO: 7 (FUS 8828:14896)

SEQ ID NO: 8 (FUS 10665:14856)

SEQ ID NO: 9 (FUS 6075:13799)

SEQ ID NO: 10 (FUS 6325:13989)

SEQ ID NO: 11 (FUS 7438:13476)

SEQ ID NO: 12 (FUS 7775:13532)

SEQ ID NO: 13 (FUS 8213:13991)

SEQ ID NO: 14 (FUS 9191:12909)

SEQ ID NO: 15 (FUS 9574:12972)

SEQ ID NO: 16 (FUS 10367:12829)

SEQ ID NO: 17(FUS 8469:13447; OrigMet)

SEQ ID NO: 51 (FUS 9144:13816), and

fragments or variants thereof.

Probes

Another aspect of the invention is to provide a hybridization probecapable of recognizing an aberrant mtDNA sequence of the invention. Asused herein, the term “probe” refers to an oligonucleotide which forms aduplex structure with a sequence in the target nucleic acid, due tocomplementarity of at least one sequence in the probe with a sequence inthe target region. The probe may be labeled, according to methods knownin the art.

Once aberrant mtDNA associated with a particular disease is identified,hybridization of mtDNA to, for example, an array of oligonucleotides canbe used to identify particular mutations, however, any known method ofhybridization may be used.

As with the primers of the present invention, probes may be generateddirectly against exemplary mtDNA fusion molecules of the invention, orto a fragment or variant thereof. For instance, the sequences set forthin SEQ ID NOs: 2-17 and 51 and those disclosed in Table 1 can be used todesign primers or probes that will detect a nucleic acid sequencecomprising a fusion sequence of interest. As would be understood bythose of skill in the art, primers or probes which hybridize to thesenucleic acid molecules may do so under highly stringent hybridizationconditions or lower stringency conditions, such conditions known tothose skilled in the art and found, for example, in Current Protocols inMolecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6.

In specific embodiments of the invention, the probes of the inventioncontain a sequence complementary to at least a portion of the aberrantmtDNA comprising the junction point of the spliced genes. This portionincludes at least one of the nucleotides involved in the junction pointA:B, and may further comprise one or more nucleotides adjacent thereto.In this regard, the present invention encompasses any suitable targetingmechanism that will select an mtDNA molecule using the nucleotidesinvolved and/or adjacent to the junction point A:B.

Various types of probes known in the art are contemplated by the presentinvention. For example, the probe may be a hybridization probe, thebinding of which to a target nucleotide sequence can be detected using ageneral DNA binding dye such as ethidium bromide, SYBR® Green, SYBR®Gold and the like. Alternatively, the probe can incorporate one or moredetectable labels. Detectable labels are molecules or moieties aproperty or characteristic of which can be detected directly orindirectly and are chosen such that the ability of the probe tohybridize with its target sequence is not affected. Methods of labellingnucleic acid sequences are well-known in the art (see, for example,Ausubel et al., (1997 & updates) Current Protocols in Molecular Biology,Wiley & Sons, New York).

Labels suitable for use with the probes of the present invention includethose that can be directly detected, such as radioisotopes,fluorophores, chemiluminophores, enzymes, colloidal particles,fluorescent microparticles, and the like. One skilled in the art willunderstand that directly detectable labels may require additionalcomponents, such as substrates, triggering reagents, light, and the liketo enable detection of the label. The present invention alsocontemplates the use of labels that are detected indirectly.

The probes of the invention are preferably at least about 15 nt, andmore preferably at least about 20 nt, still more preferably at leastabout 30 nt, and even more preferably, at least about 40 nt, at leastabout 50 nt, at least about 75 nt, or at least about 150 nt in length. Aprobe of “at least 20 nt in length,” for example, is intended to include20 or more contiguous bases that are complementary to an mtDNA sequenceof the invention. Of course, larger probes (e.g., 50, 150, 500, 600,2000 nucleotides) may be preferable.

The probes of the invention will also hybridize to nucleic acidmolecules in biological samples, thereby enabling the methods of theinvention. Accordingly, in one aspect of the invention, there isprovided a hybridization probe for use in the detection of cancer,wherein the probe is complementary to at least a portion of an aberrantmtDNA molecule. In another aspect the present invention provides probesand a use of (or a method of using) such probes for the detection ofcolorectal cancer, lung cancer, breast cancer, ovarian cancer,testicular, cancer, prostate cancer and/or melanoma skin cancer.

Assays

Measuring the level of aberrant mtDNA in a biological sample candetermine the presence of one or more cancers in a subject. The presentinvention, therefore, encompasses methods for predicting, diagnosing ormonitoring cancer, comprising obtaining one or more biological samples,extracting mtDNA from the samples, and assaying the samples for aberrantmtDNA by: quantifying the amount of one or more aberrant mtDNA sequencesin the sample and comparing the quantity detected with a referencevalue. As would be understood by those of skill in the art, thereference value is based on whether the method seeks to predict,diagnose or monitor cancer. Accordingly, the reference value may relateto mtDNA data collected from one or more known non-cancerous biologicalsamples, from one or more known cancerous biological samples, and/orfrom one or more biological samples taken over time.

In one aspect, the invention provides a method of detecting cancer in amammal, the method comprising assaying a tissue sample from the mammalfor the presence of an aberrant mitochondrial DNA described above. Thepresent invention also provides for methods comprising assaying a tissuesample from the mammal by hybridizing the sample with at least onehybridization probe. The probe may be generated against a mutantmitochondrial DNA sequence of the invention as described herein.

In another aspect, the invention provides a method as above, wherein theassay comprises:

a) conducting a hybridization reaction using at least one of the probesto allow the at least one probe to hybridize to a complementary aberrantmitochondrial DNA sequence;

b) quantifying the amount of the at least one aberrant mitochondrial DNAsequence in the sample by quantifying the amount of the mitochondrialDNA hybridized to the at least one probe; and,

c) comparing the amount of the mitochondrial DNA in the sample to atleast one known reference value.

Also included in the present invention are methods for predicting,diagnosing or monitoring cancer comprising diagnostic imaging assays asdescribed below. The diagnostic assays of the invention can be readilyadapted for high-throughput. High-throughput assays provide theadvantage of processing many samples simultaneously and significantlydecrease the time required to screen a large number of samples. Thepresent invention, therefore, contemplates the use of the nucleotides ofthe present invention in high-throughput screening or assays to detectand/or quantitate target nucleotide sequences in a plurality of testsamples.

Fusion Transcripts

The present invention further provides the identification of fusiontranscripts and associated hybridization probes useful in methods forpredicting, diagnosing and/or monitoring cancer. One of skill in the artwill appreciate that such molecules may be derived through the isolationof naturally-occurring transcripts or, alternatively, by the recombinantexpression of mtDNAs isolated according to the methods of the invention.As discussed, such mtDNAs typically comprise a spliced gene having theinitiation codon from the first gene and the termination codon of thesecond gene. Accordingly, fusion transcripts derived therefrom comprisea junction point associated with the spliced genes.

Detection of Fusion Transcripts

Naturally occurring fusion transcripts can be extracted from abiological sample and identified according to any suitable method knownin the art, or may be conducted according to the methods described inthe examples. In one embodiment of the invention, stable polyadenylatedfusion transcripts are identified using Oligo(dT) primers that targettranscripts with poly-A tails, followed by RT-PCR using primer pairsdesigned against the target transcript.

The following exemplary fusion transcripts were detected using suchmethods and found useful in predicting, diagnosing and/or monitoringcancer as indicated in the examples. Likewise, fusion transcriptsderived from the ORF sequences identified in Table 1 may be useful inpredicting, diagnosing and/or monitoring cancer according to the assaysand methods of the present invention.

SEQ ID NO: 18 (Transcripts 1;8469:13447; AltMet)

SEQ ID NO: 19 (Transcript 2;10744:14124)

SEQ ID NO: 20 (Transcript 3;7974:15496)

SEQ ID NO: 21 (Transcript 4;7992:15730)

SEQ ID NO: 22 (Transcript 5;8210:15339)

SEQ ID NO: 23 (Transcript 6;8828:14896)

SEQ ID NO: 24 (Transcript 7;10665:14856)

SEQ ID NO: 25 (Transcript 8;6075:13799)

SEQ ID NO: 26 (Transcript 9;6325:13989)

SEQ ID NO: 27 (Transcript 10;7438:13476)

SEQ ID NO: 28 (Transcript 11;7775:13532)

SEQ ID NO: 29 (Transcript 12;8213:13991)

SEQ ID NO: 30 (Transcript 14;9191:12909)

SEQ ID NO: 31 (Transcript 15;9574:12972)

SEQ ID NO: 32 (Transcript 16;10367:12829)

SEQ ID NO: 33 (Transcript 20;8469:13447; OrigMet)

SEQ ID NO: 50 (Transcript 13; 9144:13816)

Further, fusion transcripts of like character to those described hereinare contemplated for use in the field of clinical oncology.

Fusion transcripts can also be produced by recombinant techniques knownin the art. Typically this involves transformation (includingtransfection, transduction, or infection) of a suitable host cell withan expression vector comprising an mtDNA sequence of interest.

Variants or fragments of the fusion transcripts identified herein arealso provided. Such sequences may adhere to the size limitations andpercent identities described above with respect to genomic variants andfragments, or as determined suitable by a skilled technician.

In addition, putative protein sequences corresponding to transcripts1-16 and 20 are listed below. These sequences, which encode hypotheticalfusion proteins, are provided as a further embodiment of the presentinvention.

SEQ ID NO: 34 (Transcripts 1)

SEQ ID NO: 35 (Transcript 2)

SEQ ID NO: 36 (Transcript 3)

SEQ ID NO: 37 (Transcript 4)

SEQ ID NO: 38 (Transcript 5)

SEQ ID NO: 39 (Transcript 6)

SEQ ID NO: 40 (Transcript 7)

SEQ ID NO: 41 (Transcript 8)

SEQ ID NO: 42 (Transcript 9)

SEQ ID NO: 43 (Transcript 10)

SEQ ID NO: 44 (Transcript 11)

SEQ ID NO: 45 (Transcript 12)

SEQ ID NO: 46 (Transcript 14)

SEQ ID NO: 47 (Transcript 15)

SEQ ID NO: 48 (Transcript 16)

SEQ ID NO: 49 (Transcripts 20)

SEQ ID NO: 52 (Transcript 13)

Probes

Once a fusion transcript has been characterized, primers or probes canbe developed to target the transcript in a biological sample. Suchprimers and probes may be prepared using any known method (as describedabove) or as set out in the examples provided below. A probe may, forexample, be generated for the fusion transcript, and detectiontechnologies, such as QuantiGene 2.0™ by Panomics™, used to detect thepresence of the transcript in a sample. Primers and probes may begenerated directly against exemplary fusion transcripts of theinvention, or to a fragment or variant thereof. For instance, thesequences set forth in SEQ ID NOs: 18-33 and 50 as well as thosedisclosed in Table 1 can be used to design probes that will detect anucleic acid sequence comprising a fusion sequence of interest.

As would be understood by those skilled in the art, probes designed tohybridize to the fusion transcripts of the invention contain a sequencecomplementary to at least a portion of the transcript expressing thejunction point of the spliced genes. This portion includes at least oneof the nucleotides complementary to the expressed junction point, andmay further comprise one or more complementary nucleotides adjacentthereto. In this regard, the present invention encompasses any suitabletargeting mechanism that will select a fusion transcript that uses thenucleotides involved and adjacent to the junction point of the splicedgenes.

Various types of probes and methods of labelling known in the art arecontemplated for the preparation of transcript probes. Such types andmethods have been described above with respect to the detection ofgenomic sequences. The transcript probes of the invention are preferablyat least about 15 nt, and more preferably at least about 20 nt, stillmore preferably at least about 30 nt, and even more preferably, at leastabout 40 nt, at least about 50 nt, at least about 75 nt, or at leastabout 150 nt in length. A probe of “at least 20 nt in length,” forexample, is intended to include 20 or more contiguous bases that arecomplementary to an mtDNA sequence of the invention. Of course, largerprobes (e.g., 50, 150, 500, 600, 2000 nucleotides) may be preferable.

In one aspect, the invention provides a hybridization probe for use inthe detection of cancer, wherein the probe is complementary to at leasta portion of a mitochondrial fusion transcript provided above.

In another aspect, the present invention provides probes and a use of(or a method of using) such probes for the detection of colorectalcancer, lung cancer, breast cancer, ovarian cancer, testicular cancer,prostate cancer or melanoma skin cancer.

Assays

Measuring the level of mitochondrial fusion transcripts in a biologicalsample can determine the presence of one or more cancers in a subject.The present invention, therefore, provides methods for predicting,diagnosing or monitoring cancer, comprising obtaining one or morebiological samples, extracting mitochondrial RNA from the samples, andassaying the samples for fusion transcripts by: quantifying the amountof one or more fusion transcripts in the sample and comparing thequantity detected with a reference value. As would be understood bythose of skill in the art, the reference value is based on whether themethod seeks to predict, diagnose or monitor cancer. Accordingly, thereference value may relate to transcript data collected from one or moreknown non-cancerous biological samples, from one or more known cancerousbiological samples, and/or from one or more biological samples takenover time.

In one aspect, the invention provides a method of detecting a cancer ina mammal, the method comprising assaying a tissue sample from saidmammal for the presence of at least one fusion transcript of theinvention by hybridizing said sample with at least one hybridizationprobe having a nucleic acid sequence complementary to at least a portionof the mitochondrial fusion transcript.

In another aspect, the invention provides a method as above, wherein theassay comprises:

a) conducting a hybridization reaction using at least one of theabove-noted probes to allow the at least one probe to hybridize to acomplementary mitochondrial fusion transcript;

b) quantifying the amount of the at least one mitochondrial fusiontranscript in the sample by quantifying the amount of the transcripthybridized to the at least one probe; and,

c) comparing the amount of the mitochondrial fusion transcript in thesample to at least one known reference value.

As discussed above, the diagnostic assays of the invention may alsocomprise diagnostic methods and screening tools as described herein andcan be readily adapted for high-throughput. The present invention,therefore, contemplates the use of the fusion transcripts and associatedprobes of the present invention in high-throughput screening or assaysto detect and/or quantitate target nucleotide sequences in a pluralityof test samples.

Diagnostic Methods and Screening Tools

Methods and screening tools for diagnosing specific diseases oridentifying specific mitochondrial mutations are also hereincontemplated. Any known method of hybridization may be used to carry outsuch methods including, without limitation, probe/primer basedtechnologies such as branched DNA and qPCR, both single-plex andmulti-plex. Array technology, which has oligonucleotide probes matchingthe wild type or mutated region, and a control probe, may also be used.Commercially available arrays such as microarrays or gene chips aresuitable. These arrays contain thousands of matched and control pairs ofprobes on a slide or microchip, and are capable of sequencing the entiregenome very quickly. Review articles describing the use of microarraysin genome and DNA sequence analysis are available on-line.

Screening tools designed to identify targets which are relevant to agiven biological condition may include specific arrangements of nucleicacids associated with a particular disease or disorder. Thus, inaccordance with one embodiment of the invention, there is provided ascreening tool comprised of a microarray having 10's, 100's, or 1000'sof mitochondrial fusion transcripts for identification of thoseassociated with one or more cancers. In accordance with anotherembodiment, there is provided a screening tool comprised of a microarrayhaving 10's, 100's, or 1000's of mitochondrial DNAs corresponding tomitochondrial fusion transcripts for identification of those associatedwith one or more cancers. In a further embodiment, there is provided ascreening tool comprised of a multiplexed branched DNA assay having10's, 100's, or 1000's of mitochondrial fusion transcripts foridentification of those associated with one or more cancers. In yetanother embodiment of the invention, there is provided a screening toolcomprised of a multiplexed branched DNA assay having 10's, 100's, or1000's of mitochondrial DNAs corresponding to mitochondrial fusiontranscripts for identification of those associated with one or morecancers.

Approaches useful in the field of clinical oncology are also hereincontemplated and may include such diagnostic imaging techniques asPositron Emission Tomography (PET), contrast Magnetic Resonance Imaging(MRI) or the like. These diagnostic methods are well known to those ofskill in the art and are useful in the diagnosis and prognosis ofcancer.

Diagnostic Monitoring

The methods of the present invention may further comprise the step ofrecommending a monitoring regime or course of therapy based on theoutcome of one or more assays. This allows clinicians to practicepersonalized medicine; e.g. cancer therapy, by monitoring theprogression of the patient's cancer (such as by recognizing when aninitial or subsequent mutation occurs) or treatment (such as byrecognizing when a mutation is stabilized).

With knowledge of the boundaries of the sequence variation in hand, theinformation can be used to diagnose a pre-cancerous condition orexisting cancer condition. Further, by quantitating the amount ofaberrant mtDNA in successive samples over time, the progression of acancer condition can be monitored. For example, data provided byassaying the patient's tissues at one point in time to detect a firstset of mutations from wild-type could be compared against data providedfrom a subsequent assay, to determine if changes in the aberration haveoccurred.

Where a mutation is found in an individual who has not yet developedsymptoms of cancer, the mutation may be indicative of a geneticsusceptibility to develop a cancer condition. A determination ofsusceptibility to disease or diagnosis of its presence can further beevaluated on a qualitative basis based on information concerning theprevalence, if any, of the cancer condition in the patient's familyhistory and the presence of other risk factors, such as exposure toenvironmental factors and whether the patient's cells also carry amutation of another sort.

Biological Sample

The present invention provides for diagnostic tests which involveobtaining or collecting one or more biological samples. In the contextof the present invention, “biological sample” refers to a tissue orbodily fluid containing cells from which mtDNA and mtRNA can beobtained. For example, the biological sample can be derived from tissueincluding, but not limited to, skin, lung, breast, prostate, nervous,muscle, heart, stomach, colon, rectal tissue and the like; or fromblood, saliva, cerebral spinal fluid, sputa, urine, mucous, synovialfluid, peritoneal fluid, amniotic fluid and the like. The biologicalsample may be obtained from a cancerous or non-cancerous tissue and maybe, but is not limited to, a surgical specimen or a biopsy specimen.

The biological sample can be used either directly as obtained from thesource or following a pre-treatment to modify the character of thesample. Thus, the biological sample can be pre-treated prior to use by,for example, preparing plasma or serum from blood, disrupting cells,preparing liquids from solid materials, diluting viscous fluids,filtering liquids, distilling liquids, concentrating liquids,inactivating interfering components, adding reagents, and the like.

One skilled in the art will understand that more than one sample typemay be assayed at a single time (i.e. for the detection of more than onecancer). Furthermore, where a course of collections are required, forexample, for the monitoring of cancer over time, a given sample may bediagnosed alone or together with other samples taken throughout a testperiod. In this regard, biological samples may be taken once only, or atregular intervals such as biweekly, monthly, semi-annually or annually.

Kits

The present invention provides diagnostic/screening kits for detectingcancer in a clinical environment. Such kits may include one or moresampling means, in combination with one or more probes according to thepresent invention.

The kits can optionally include reagents required to conduct adiagnostic assay, such as buffers, salts, detection reagents, and thelike. Other components, such as buffers and solutions for the isolationand/or treatment of a biological sample, may also be included in thekit. One or more of the components of the kit may be lyophilised and thekit may further comprise reagents suitable for the reconstitution of thelyophilised components.

Where appropriate, the kit may also contain reaction vessels, mixingvessels and other components that facilitate the preparation of the testsample. The kit may also optionally include instructions for use, whichmay be provided in paper form or in computer-readable form, such as adisc, CD, DVD or the like.

In one embodiment of the invention there is provided a kit fordiagnosing cancer comprising sampling means and a hybridization probe ofthe invention.

Various aspects of the invention will be described by illustration usingthe following examples. The examples provided herein serve only toillustrate certain specific embodiments of the invention and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Detection of Mitochondrial Fusion Transcripts

The mitochondrial 4977 “common deletion” and a 3.4 kb deletionpreviously identified by the present Applicant in PCT application no.PCT/CA2007/001711 (the entire contents of which are incorporated byreference) result in unique open reading frames having activetranscripts as identified by oligo-dT selection in prostate tissue(FIGS. 2 and 3). Examination of breast tissue samples also reveals thepresence of a stable polyadenylated fusion transcript resulting from the3.4 kb deletion (FIG. 4).

Reverse Transcriptase-PCR Protocol for Deletion Transcript Detection

RNA Isolation cDNA Synthesis

Total RNA was isolated from snap frozen prostate and breast tissuesamples (both malignant and normal samples adjacent to tumours) usingthe Aurum™ Total RNA Fatty and Fibrous Tissue kit (Bio-Rad, Hercules,Calif.) following the manufacturer's instructions. Since in thisexperiment, genomic DNA contamination was to be avoided, a DNase Itreatment step was included, using methods as commonly known in the art.RNA quantity and quality were determined with an ND-1000spectrophotometer (NanoDrop® technologies). From a starting material ofabout 100 g, total RNA concentrations varied from 100-1000 ng/ul with a260/280 ratio between 1.89-2.10. RNA concentrations were adjusted to 100ng/ul and 2 ul of each template were used for first strand DNA synthesiswith SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen)following the manufacturer's instructions. In order to identify stablepolyadenylated fusion transcripts, Oligo(dT) primers that targettranscripts with poly-A tails were used.

PCR

Real time PCR was performed using 5 ul of each cDNA template with theiQ™ SYBR® Green Supermix (Bio-Rad, Hercules, Calif.) on DNA EngineOpticon® 2 Continuous Fluorescence Detection System (Bio-Rad, Hercules,Calif.). The primer pairs targeting the 4977 bp deletion are; 8416F5′-CCTTACACTATTCCTCATCAC-3′, 13637R 5′-TGACCTGTTAGGGTGAGAAG-3′, andthose for the 3.4 kb deletion are; ND4LF 5′-TCGCTCACACCTCATATCCTC-3′,ND5R 5′-TGTGATTAGGAGTAGGGTTAGG-3′. The reaction cocktail included: 2×SYBR® Green Supermix (100 mM KCL, 40 mM Tris-HCl, pH 8.4, 0.4 mM of eachdNTP [dATP, dCTP, dGTP, and dTTP], iTaq™ DNA polymerase, 50 units/ml, 6mM MgCl₂, SYBR® Green 1, 20 nM flourescein, and stabilizers), 250 nMeach of primers, and ddH₂O. PCR cycling parameters were as follows; (1)95° C. for 2 min, (2) 95° C. for 30 sec, (3) 55° C. (for the 4977 bpdeletion) and 63° C. (for the 3.4 kb deletion) for 30 sec, (4) 72° C.for 45 sec, (5) plate read, followed by 39 cycles of steps 3 to 5, andfinal incubation at 4° C. Apart from cycling threshold and melting curveanalysis, samples were run on agarose gels for specific visualization ofamplification products (see FIGS. 2 to 4).

FIG. 2 is an agarose gel showing polyadenalated fusion transcripts inprostate samples invoked by the loss of 3.4 kb from the mitochondrialgenome. Legend for FIG. 2: B-blank, Lanes 1-6 transcripts detected incDNA; lanes 7-12 no reverse transcriptase (RT) controls for samples inlanes 1-6.

FIG. 3 shows polyadenalated fusion transcripts in prostate samplesinvoked by the loss of the 4977 kb common deletion. Legend for FIG. 3:B-blank, Lanes 1-6 transcripts detected in cDNA; lanes 7-12 no RTcontrols for samples in lanes 1-6.

FIG. 4 shows polyadenalated fusion transcripts in breast samples invokedby the loss of 3.4 kb from the mtgenome. Legend for FIG. 4: Lanes 2-8transcripts from breast cDNAs; lane 9 negative (water) control; lanes 10and 11, negative, no RT, controls for samples in lanes 2 and 3.

These results demonstrate the existence of stable mitochondrial fusiontranscripts.

Example 2 Identification and Targetting of Fusion Products

Various hybridization probes were designed to detect, and furtherdemonstrate the presence of novel transcripts resulting from mutatedmitochondrial genomes, such as the 3.4 kb deletion. For this purpose, asingle-plex branched DNA platform for quantitative gene expressionanalysis (QuantiGene 2.0™, Panomics™) was utilized. The specificdeletions and sequences listed in this example are based on theirrelative positions with the entire mtDNA genome, which is recited in SEQID NO: 1. The nucleic acid sequences of the four transcript to which theprobes were designed in this example are identified herein as follows:Transcript 1 (SEQ ID NO: 18), Transcript 2 (SEQ ID NO: 19), Transcript 3(SEQ ID NO: 20) and Transcript 4 (SEQ ID NO: 21).

An example of a continuous transcript from the 3.4 kb mitochondrialgenome deletion occurs with the genes ND4L (NADH dehydrogenase subunit4L) and ND5 (NADH dehydrogenase subunit 5). A probe having acomplementary sequence to SEQ ID NO: 19, was used to detect transcript2. The repetitive elements occur at positions 10745-10754 in ND4L and14124-14133 in ND5.

The 3.4 kb deletion results in the removal of the 3′ end of ND4L, thefull ND4 gene, tRNA histidine, tRNA serine2, tRNA leucine2, and themajority of the 5′ end of ND5 (see FIG. 5 a), resulting in a gene spliceof ND4L and ND5 with a junction point of 10744(ND4L):14124(ND5) (FIG. 5b). SEQ ID NO: 3 is the complementary DNA sequence to the RNA transcript(SEQ ID NO: 19) detected in the manner described above.

Similarly, transcript 1 is a fusion transcript between ATPase 8 and ND5associated with positions 8469:13447 (SEQ ID NO: 18). Transcripts 3 and4 (SEQ ID NO: 20 and SEQ ID NO: 21, respectively) are fusion transcriptsbetween COII and Cytb associated with nucleotide positions 7974:15496and 7992:15730 respectively. Table 3 provides a summary of therelationships between the various sequences used in this example. Table3 includes the detected fusion transcript and the DNA sequencecomplementary to the fusion transcript detected.

Example 3 Application to Prostate Cancer

Using the four fusion transcripts, i.e. transcripts 1 to 4, discussedabove, two prostate tissue samples from one patient were analyzed toassess the quantitative difference of the novel predicted fusiontranscripts. The results of the experiment are provided in Table 2below, wherein “Homog 1” refers to the homogenate of frozen prostatetumour tissue from a patient and “Homog 2” refers to the homogenate offrozen normal prostate tissue adjacent to the tumour of the patient.These samples were processed according to the manufacturer's protocol(QuantiGene® Sample Processing Kit for Fresh or Frozen Animal Tissues;and QuantiGene® 2.0 Reagent System User Manual) starting with 25.8 mg ofHomog 1 and 28.9 mg of Homog 2 (the assay setup is shown in Tables 5aand 5b).

Clearly demonstrated is an increased presence of mitochondrial fusiontranscripts in prostate cancer tissue compared to normal adjacentprostate tissue. The fusion transcript is present in the normal tissue,although at much lower levels. The relative luminescence units (RLU)generated by hybridization of a probe to a target transcript aredirectly proportional to the abundance of each transcript. Table 2 alsoindicates the coefficients of variation, CV, expressed as a percentage,of the readings taken for the samples. The CV comprises the Standarddeviation divided by the average of the values. The significance of suchstably transcribed mitochondrial gene products in cancer tissue hasimplications in disease evolution and progression.

Example 4 Application to Breast Cancer

Using the same protocol from Example 3 but focusing only on Transcript2, the novel fusion transcript associated with the 3.4 kb mtgenomedeletion, analyses were conducted on two samples of breast tumour tissueand two samples of tumour-free tissues adjacent to those tumours, aswell as three samples of prostate tumour tissue, one sample comprisingadjacent tumour-free tissue. Results for this example are provided inTable 4. The prostate tumour tissue sample having a corresponding normaltissue section demonstrated a similar pattern to the prostate sampleanalyzed in Example 3 in that the tumour tissue had approximately 2times the amount of the fusion transcript than did the normal adjacenttissue. The breast tumour samples demonstrated a marked increase in thefusion transcript levels when compared to the adjacent non-tumourtissues. A 1:100 dilution of the homogenate was used for this analysisas it performed most reproducibly in the experiment cited in Example 3.

Thus, the above discussed results illustrate the application of thetranscripts of the invention in the detection of tumours of bothprostate and breast tissue.

Example 5 Application to Colorectal Cancer

This study sought to determine the effectiveness of several transcriptsof the invention in detecting colorectal cancer. A total of 19 sampleswere prepared comprising nine control (benign) tissue samples (samples 1to 9) and ten tumour (malignant) tissue samples (samples 10 to 19). Thesamples were homogenized according to the manufacturer's recommendations(Quantigene® Sample Processing Kit for Fresh or Frozen Animal Tissues;and Quantigene 2.0 Reagent System User Manual). Seven target transcriptsand one housekeeper transcript were prepared in the manner as outlinedabove in previous examples. The characteristics of the transcripts aresummarized as follows:

TABLE 7 Characteristics of Breast Cancer Transcripts Transcript IDJunction Site Gene Junction 2 10744:14124  ND4L:ND5 3 7974:15496COII:Cytb 10 7438:13476 COI:ND5 11 7775:13532 COII:ND5 12 8213:13991COII:ND5 Peptidylpropyl isomerase B (PPIB) N/A N/A (“housekeeper”)

It is noted that transcripts 2 and 3 are the same as those discussedabove with respect to Examples 3 and 4.

Homogenates were prepared using approximately 25 mg of tissue from OCTblocks and diluted 1:1 for transcripts 2 and 4, and 1:8 for transcripts10 and 11. The quantity of the transcripts was measured in RelativeLuminenscence Units RLU on a Glomax™ Multi Detection System (Promega).All samples were assayed in triplicate for each transcript. Backgroundmeasurements (no template) were done in triplicate as well. The analysisaccounted for background by subtracting the lower limit from the RLUvalues for the samples. Input RNA was accounted for by using the formulalog₂ a RLU−log₂ h RLU where a is the target fusion transcript and h isthe housekeeper transcript.

The analysis of the data comprised the following steps:

a) Establish CV's (coefficients of variation) for triplicate assays;acceptable if 15%.

b) Establish average RLU value for triplicate assays of target fusiontranscript(a) and housekeeper transcript (h).

c) Establish lower limit from triplicate value of background RLU (l).

d) Subtract lower limit (l) from (a).

e) Calculate log₂ a RLU−log₂ h RLU.

Summary of Results:

The results of the above analysis are illustrated in FIGS. 6 a to 6 g,which comprise plots of the log₂ a RLU−log₂ h RLU against sample number.Also illustrated are the respective ROC (Receiver OperatingCharacteristic) curves determined from the results for each transcript.

Transcript 2: There exists a statistically significant differencebetween the means (p<0.10) of the normal and malignant groups (p>0.09),using a cutoff value of 3.6129 as demonstrated by the ROC curve resultsin a sensitivity of 60% and specificity of 89% and the area under thecurve is 0.73 indicating fair test accuracy. The threshold value chosenmay be adjusted to increase either the specificity or sensitivity of thetest for a particular application.

Transcript 3: There exists a statistically significant differencebetween the means (p<0.05) of the normal and malignant groups (p=0.03),using a cutoff value of 4.0813 as demonstrated by the ROC curve resultsin a sensitivity of 60% and specificity of 78% and the area under thecurve is 0.79 indicating fair to good test accuracy. The threshold valuechosen may be adjusted to increase either the specificity or sensitivityof the test for a particular application.

Transcript 8: There exists a statistically significant differencebetween the means (p<0.1) of the normal and malignant groups (p=0.06).Using a cutoff value of −6.0975 as demonstrated by the ROC curve resultsin a sensitivity of 60% and specificity of 89% and the area under thecurve is 0.76 indicating fair test accuracy. The threshold value chosenmay be adjusted to increase either the specificity or sensitivity of thetest for a particular application.

Transcript 9: There exists a statistically significant differencebetween the means (p<0.1) of the normal and malignant groups (p=0.06).Using a cutoff value of −7.5555 as demonstrated by the ROC curve resultsin a sensitivity of 60% and specificity of 89% and the area under thecurve is 0.76 indicating fair to good test accuracy. The threshold valuechosen may be adjusted to increase either the specificity or sensitivityof the test for a particular application.

Transcript 10: There is a statistically significant difference betweenthe means (p≦0.01) of the normal and malignant groups (p=0.01). Using acutoff value of −3.8272 as demonstrated by the ROC curve results in asensitivity of 90% and specificity of 67% and the area under the curveis 0.84, indicating good test accuracy. The threshold value chosen maybe adjusted to increase either the specificity or sensitivity of thetest for a particular application.

Transcript 11: There exists a statistically significant differencebetween the means (p<0.1) of the normal and malignant groups (p=0.06),using a cutoff value of 3.1753 as demonstrated by the ROC curve resultsin a sensitivity of 70% and specificity of 78% and the area under thecurve is 0.76 indicating fair to good test accuracy. The threshold valuechosen may be adjusted to increase either the specificity or sensitivityof the test for a particular application.

Transcript 12: There exists a statistically significant differencebetween the means (p<0.1) of the normal and malignant groups (p=0.06),using a cut-off value of 3.2626 as demonstrated by the ROC curve resultsin a sensitivity of 70% and specificity of 78% and the area under thecurve is 0.76 indicating fair to good test accuracy. The threshold valuechosen may be adjusted to increase either the specificity or sensitivityof the test for a particular application.

Conclusions:

The above results illustrate the utility of transcripts 2, 3, 8, 9, 10,11, and 12 in the detection of colorectal cancer and in distinguishingmalignant from normal colorectal tissue. As indicated above, transcripts2 and 3 were also found to have utility in the detection of prostatecancer. Transcript 2 was also found to have utility in the detection ofbreast cancer. Transcript 11 was also found to have utility in thedetection of melanoma skin cancer. Transcript 10 was also found to haveutility in the detection of lung cancer and melanoma. Transcript 8 wasalso found to have utility in the detection of lung cancer. Any of the 7transcripts listed may be used individually or in combination as a toolfor the detection of characterization of colorectal cancer in a clinicalsetting.

Example 6 Application to Lung Cancer

This study sought to determine the effectiveness of several transcriptsof the invention in the detection of lung cancer. As in Example 5, ninecontrol (benign) tissue samples (samples 1 to 9) and ten tumour(malignant) tissue samples (samples 10 to 19) were homogenized accordingto the manufacturer's recommendations (Quantigene® Sample Processing Kitfor Fresh or Frozen Animal Tissues; and Quantigene 2.0 Reagent SystemUser Manual). Homogenates were diluted 1:8 and the quantity of 4 targettranscripts and 1 housekeeper transcript was measured in RelativeLuminenscence Units RLU on a Glomax™ Multi Detection System (Promega).All samples were assayed in triplicate for each transcript. Backgroundmeasurements (no template) were done in triplicate as well.

The following transcripts were prepared for this example:

TABLE 8 Characteristics of Lung Cancer Transcripts Transcript IDJunction Site Gene Junction 6 8828:14896 ATPase6:Cytb 8 6075:13799COI:ND5 10 7438:13476 COI:ND5 20 8469:13447 ATPase8:ND5 Peptidylpropylisomerase B (PPIB) N/A N/A (“housekeeper”)

The tissue samples used in this example had the followingcharacteristics:

TABLE 9 Characteristics of Lung Cancer Samples Sample Malignant Comments(source of tissue) 1 NO interstitial lung disease 2 NO emphysema 3 NOaneurysm 4 NO bronchopneumonia, COPD 5 NO malignant neoplasm in liver,origin unknown, calcified granulomas in lung 6 NO 12 hours post mortem,mild emphysema 7 NO 12 hours post mortem, large B cell lymphoma,pulmonary edema, pneumonia 8 NO pneumonia, edema, alveolar damage 9 NOcongestion and edema 10 YES adenocarcinoma, non-small cell 11 YES smallcell 12 YES squamous cell carcinoma, NSC, emphysema 13 YESadenocarcinoma, lung cancer, nsc, metastatic 14 YES squamous cellcarcinoma, non-small cell 15 YES mixed squamous and adenocarcinoma 16YES non-small cell carcinoma, squamous 17 YES small cell carcinoma 18YES adenocarcinoma, lung cancer, nsc 19 YES adenocarcinoma, lung cancer,nsc, metastatic

The analysis of data was performed according to the method described inExample 5. The results are illustrated in FIGS. 7 a, 7 b, 7 c and 7 d.

Summary of Results:

Transcript 6: There exists a statistically significant differencebetween the means (p<0.1) of the normal (benign) and malignant groups(p=0.06), using a cutoff value of −6.5691 as demonstrated by the ROCcurve results in a sensitivity of 80% and specificity of 71% and thearea under the curve is 0.77, indicating fair test accuracy. Thethreshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 8: The difference between the means of the normal andmalignant groups is statistically significant, p<0.05 (p=0.02). Using acutoff value of −9.6166 as demonstrated by the ROC curve results in asensitivity of 90% and specificity of 86% and the area under the curveis 0.86 indicating good test accuracy. The threshold value chosen may beadjusted to increase either the specificity or sensitivity of the testfor a particular application.

Transcript 10: The difference between the means of the normal andmalignant groups is statistically significant, p≦0.01 (p=0.01). Using acutoff value of −10.6717 as demonstrated by the ROC curve results in asensitivity of 90% and specificity of 86% and the area under the curveis 0.89 indicating good test accuracy. The threshold value chosen may beadjusted to increase either the specificity or sensitivity of the testfor a particular application.

Transcript 20: The difference between the means of the normal andmalignant groups is statistically significant, p≦0.1 (p=0.1). Using acutoff value of 2.5071 as demonstrated by the ROC curve results in asensitivity of 70% and specificity of 71% and the area under the curveis 0.74 indicating fair test accuracy. The threshold value chosen may beadjusted to increase either the specificity or sensitivity of the testfor a particular application.

Conclusions:

The results from example 6 illustrate the utility of transcripts 6, 8,10, and 20 of the invention in the detection of lung cancer tumours andthe distinction between malignant and normal lung tissues. Any of thesethree transcripts may be used for the detection or characterization oflung cancer in a clinical setting.

Example 7 Application to Melanoma

This study sought to determine the effectiveness of several transcriptsof the invention in the detection of melanomas. In this study a total of14 samples were used, comprising five control (benign) tissue samplesand nine malignant tissue samples. All samples were formalin fixed,paraffin embedded (FFPE). The FFPE tissue samples were sectioned intotubes and homogenized according to the manufacturer's recommendations(Quantigene® 2.0 Sample Processing Kit for FFPE Samples; and Quantigene2.0 Reagent System User Manual) such that each sample approximated 20microns prior to homogenization. Homogenates were diluted 1:4 and thequantity of 7 target transcripts and 1 housekeeper transcript wasmeasured in Relative Luminenscence Units RLU on a Glomax™ MultiDetection System (Promega). All samples were assayed in triplicate foreach transcript. Background measurements (no template) were done intriplicate as well.

The 14 tissue samples used in this example had the followingcharacteristics:

TABLE 10 Characteristics of Melanoma Cancer Samples Sample MalignantComments (source of tissue) 1 NO breast reduction tissue (skin) 2 NObreast reduction tissue (skin) 3 NO breast reduction tissue (skin) 4 NObreast reduction tissue (skin) 5 NO breast reduction tissue (skin) 6 YESlentigo maligna, (melanoma in situ) invasive melanoma not present 7 YESinvasive malignant melanoma 8 YES nodular melanoma, pT3b, associatedfeatures of lentigo maligna 9 YES residual superficial spreadinginvasive malignant melanoma, Clark's level II 10 YES superficialspreading malignant melanoma, Clark's Level II 11 YES nodular malignantmelanoma, Clark's level IV 12 YES superficial spreading malignantmelanoma in situ, no evidence of invasion 13 YES superficial spreadingmalignant melanoma, Clark's level II, focally present vertical phase 14YES superficial spreading malignant melanoma in situ, Clark's level I

The following transcripts were prepared for this example:

TABLE 11 Characteristics of Melanoma Cancer Transcripts Transcript IDJunction Site GeneJunction 6 8828:4896  ATPase6:Cytb 10 7438:13476COI:ND5 11 7775:13532 COII:ND5 14 9191:12909 ATPase6:ND5 15 9574:12972COIII:ND5 16 10367:12829  ND3:ND5 20 8469:13447 ATPase8:ND5Peptidylpropyl isomerase B (PPIB) N/A N/A (“housekeeper”)

As indicated, transcripts 10 and 11 were also used in Example 5. Theanalysis of data was performed according to the method described inExample 5. The results are illustrated in FIGS. 8 a-8 g.

Summary of Results:

Transcript 6: There exists a statistically significant differencebetween the means (p≦0.01) of the normal and malignant groups (p=0.01).Further, using a cutoff value of −5.9531 as demonstrated by the ROCcurve results in a sensitivity of 89% and specificity of 80% and thearea under the curve is 0.96, indicating very good test accuracy. Thethreshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 10: There exists a statistically significant differencebetween the means (p≦0.05) of the normal and malignant groups (p=0.05),using a cutoff value of −4.7572 as demonstrated by the ROC curve resultsin a sensitivity of 89% and specificity of 40% and the area under thecurve is 0.82, indicating good test accuracy. The threshold value chosenmay be adjusted to increase either the specificity or sensitivity of thetest for a particular application.

Transcript 11: There exists a statistically significant differencebetween the means (p<0.05) of the normal and malignant groups (p=0.02).Further, using a cutoff value of 1.6762 as demonstrated by the ROC curveresults in a sensitivity of 78% and specificity of 100% and the areaunder the curve is 0.89, indicating good test accuracy. The thresholdvalue chosen may be adjusted to increase either the specificity orsensitivity of the test for a particular application.

Transcript 14: There exists a statistically significant differencebetween the means (p≦0.05) of the normal and malignant groups (p=0.05).Further, using a cutoff value of −4.9118 as demonstrated by the ROCcurve results in a sensitivity of 89% and specificity of 60% and thearea under the curve is 0.82, indicating good test accuracy. Thethreshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 15: There exists a statistically significant differencebetween the means (p<0.1) of the normal and malignant groups (p=0.07),using a cutoff value of −7.3107 as demonstrated by the ROC curve resultsin a sensitivity of 100% and specificity of 67% and the area under thecurve is 0.80, indicating good test accuracy. The threshold value chosenmay be adjusted to increase either the specificity or sensitivity of thetest for a particular application.

Transcript 16: There exists a statistically significant differencebetween the means (p<0.05) of the normal and malignant groups (p=0.03).Further, using a cutoff value of −10.5963 as demonstrated by the ROCcurve results in a sensitivity of 89% and specificity of 80% and thearea under the curve is 0.878, indicating good test accuracy. Thethreshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 20: There exists a statistically significant differencebetween the means (p<0.05) of the normal and malignant groups (p=0.04).Further, using a cutoff value of −8.3543 as demonstrated by the ROCcurve results in a sensitivity of 100% and specificity of 80% and thearea under the curve is 0.89, indicating good test accuracy. Thethreshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Conclusions:

The results from example 7 illustrate the utility of transcripts 6, 10,11, 14, 15, 16 and 20 of the invention in the detection of malignantmelanomas. As indicated above, transcripts 10 and 11 were also foundhave utility in detecting colorectal cancer while transcript 6 hasutility in the detection of lung cancer. A transcript summary by diseaseis provided at Table 6.

Example 8 Application to Ovarian Cancer

This study sought to determine the effectiveness of several transcriptsof the invention in detecting ovarian cancer. A total of 20 samples wereprepared comprising ten control (benign) tissue samples (samples 1 to10) and ten tumour (malignant) tissue samples (samples 11 to 20). Thesamples were homogenized according to the manufacturer's recommendations(Quantigene® Sample Processing Kit for Fresh or Frozen Animal Tissues;and Quantigene 2.0 Reagent System User Manual). Eight target transcriptsand one housekeeper transcript were prepared in the manner as outlinedabove in previous examples.

The 20 tissue samples used in this example had the followingcharacteristics:

TABLE 12 Characteristics of Ovarian Cancer Samples Sample DiagnosisComments 1 Normal follicular cyst 2 Normal fibroma 3 Normal Nopathological change in ovaries 4 Normal follicular cysts 5 Normalcellular fibroma 6 Normal benign follicular and simple cysts 7 Normalleiomyomata, corpora albicantia 8 Normal copora albicantia and anepithelial inclusions cysts 9 Normal corpora albicantia 10 Normalcorpora albicantia, surface inclusion cysts, follicullar cysts 11Malignant high grade poorly differentiated papillary serous carcinomainvolving omentum 12 Malignant endometrioid adenocarcinoma, well tomoderately differentiated with focal serous differentiation 13 Malignantpapillary serous carcinoma 14 Malignant mixed epithelial carcinomapredominantly papillary serous carcinoma 15 Malignant High grade: serouscarcinoma, papillary and solid growth patterns 16 Malignant High Grade(3/3) Papillary serous carcinoma 17 Malignant papillary serouscarcinoma, high nuclear grade 18 Malignant Papillary serouscystadenocarcinomas Grade: III 19 Malignant poorly differentiatedpapillary serous carcinoma 20 Malignant Well-differentiatedadnocarcinoma, Endometrioid type, Grade 1

The characteristics of the transcripts are summarized as follows:

TABLE 13 Characteristics of Ovarian Cancer Transcripts Transcript IDJunction Site Gene Junction 1 8469:13447 ATPase8:ND5 2 10744:14124 ND4L:ND5 3 7974:15496 COII:Cytb 6 8828:14896 ATPase6:Cytb 11 7775:13532COII:ND5 12 8213:13991 COII:ND5 15 9574:12972 COIII:ND5 20 8469:13447ATPase8:ND5 Ribosomal Protein Large PO (LRP) N/A N/A Housekeeper

It is noted that transcripts 1, 2, 3, 6, 11, 12, 15 and 20 are the sameas those discussed above with respect to Examples 3-7.

Homogenates were prepared using approximately 25 mg of frozen tissue anddiluted 1:4. The quantity of the transcripts was measured in RelativeLuminenscence Units RLU on a Glomax™ Multi Detection System (Promega).All samples were assayed in triplicate for each transcript. Backgroundmeasurements (no template) were done in triplicate as well. The analysisaccounted for background by subtracting the lower limit from the RLUvalues for the samples. Input RNA was accounted for by using the formulalog₂ a RLU−log₂ h RLU where a is the target fusion transcript and h isthe housekeeper transcript.

The analysis of the data comprised the following steps:

a) Establish CV's (coefficients of variation) for triplicate assays;acceptable if 15%.

b) Establish average RLU value for triplicate assays of target fusiontranscript(a) and housekeeper transcript (h).

c) Establish lower limit from triplicate value of background RLU (l).

d) Subtract lower limit (l) from (a).

e) Calculate log₂ a RLU−log₂ h RLU.

Summary of Results:

The results of the above analysis are illustrated in FIGS. 9 a to 9 h,which comprise plots of the log₂ a RLU−log₂ h RLU against sample number.Also illustrated are the respective ROC (Receiver OperatingCharacteristic) curves determined from the results for each transcript.

Transcript 1: There exists a statistically significant differencebetween the means (p<0.05) of the normal and malignant groups (p=0.002).Using a cutoff value of −11.1503 as demonstrated by the ROC curveresults in a sensitivity of 90% and specificity of 80% and the areaunder the curve is 0.91 indicating very good test accuracy. Thethreshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 2: There exists a statistically significant differencebetween the means (p<0.01) of the normal and malignant groups (p=0.001).Using a cutoff value of 0.6962 as demonstrated by the ROC curve resultsin a sensitivity of 90% and specificity of 100% and the area under thecurve is 0.96 indicating very good test accuracy. The threshold valuechosen may be adjusted to increase either the specificity or sensitivityof the test for a particular application.

Transcript 3: There exists a statistically significant differencebetween the means (p<0.01) of the normal and malignant groups (p=0.000).Using a cutoff value of 0.6754 as demonstrated by the ROC curve resultsin a sensitivity of 100% and specificity of 100% and the area under thecurve is 1.00 indicating excellent test accuracy. The threshold valuechosen may be adjusted to increase either the specificity or sensitivityof the test for a particular application.

Transcript 6: There exists a statistically significant differencebetween the means (p<0.01) of the normal and malignant groups (p=0.007).Using a cutoff value of −9.6479 as demonstrated by the ROC curve resultsin a sensitivity of 90% and specificity of 70% and the area under thecurve is 0.86 indicating good test accuracy. The threshold value chosenmay be adjusted to increase either the specificity or sensitivity of thetest for a particular application.

Transcript 11: There is a statistically significant difference betweenthe means (p<0.01) of the normal and malignant groups (p=0.000). Using acutoff value of −1.3794 demonstrated by the ROC curve results in asensitivity of 100% and specificity of 90% and the area under the curveis 0.99, indicating excellent test accuracy. The threshold value chosenmay be adjusted to increase either the specificity or sensitivity of thetest for a particular application.

Transcript 12: There exists a statistically significant differencebetween the means (p<0.01) of the normal and malignant groups (p=0.001).Using a cutoff value of −1.2379 as demonstrated by the ROC curve resultsin a sensitivity of 90% and specificity of 100% and the area under thecurve is 0.96 indicating excellent test accuracy. The threshold valuechosen may be adjusted to increase either the specificity or sensitivityof the test for a particular application.

Transcript 15: There exists a statistically significant differencebetween the means (p<0.05) of the normal and malignant groups (p=0.023).Using a cut-off value of −8.6926 as demonstrated by the ROC curveresults in a sensitivity of 70% and specificity of 80% and the areaunder the curve is 0.80 indicating good test accuracy. The thresholdvalue chosen may be adjusted to increase either the specificity orsensitivity of the test for a particular application.

Transcript 20: There exists a statistically significant differencebetween the means (p<0.01) of the normal and malignant groups (p=0.000).Using a cut-off value of 0.6521 as demonstrated by the ROC curve resultsin a sensitivity of 100% and specificity of 100% and the area under thecurve is 0.76 indicating fair to good test accuracy. The threshold valuechosen may be adjusted to increase either the specificity or sensitivityof the test for a particular application.

Conclusions:

The above results illustrate the utility of transcripts 1, 2, 3, 6, 11,12, 15, and 20 in the detection of ovarian cancer and in distinguishingmalignant from normal ovarian tissue. Transcripts 1, 2 and 3 were alsofound to have utility in the detection of prostate cancer. Transcript 6was also found to have utility in the detection of melanoma and lungcancer. Transcript 11 was also found to have utility in the detection ofmelanoma skin cancer, colorectal cancer and testicular cancer.Transcript 12 was also found to have utility in the detection ofcolorectal cancer and testicular cancer. Transcript 15 was also found tohave utility in the detection of melanoma and testicular cancer.Transcript 20 was also found to have utility in the detection ofcolorectal cancer, melanoma, and testicular cancer. Any of the 8transcripts listed may be used individually or in combination as a toolfor the detection or characterization of ovarian cancer in a clinicalsetting.

Example 9 Application to Testicular Cancer

This study sought to determine the effectiveness of several transcriptsof the invention in detecting testicular cancer. A total of 17 sampleswere prepared comprising eight control (benign) tissue samples (samples1 to 8) and 9 tumour (malignant) tissue samples (samples 9 to 17), 5 ofthe malignant samples were non-seminomas (samples 9-13) and 4 wereseminomas (samples 14-17). The samples were homogenized according to themanufacturer's recommendations (Quantigene® Sample Processing Kit forFresh or Frozen Animal Tissues; and Quantigene 2.0 Reagent System UserManual). 10 target transcripts and one housekeeper transcript wereprepared in the manner as outlined above in previous examples.

The 17 tissue samples used in this example had the followingcharacteristics:

TABLE 14 Characteristics of Testicular Cancer Samples General StratifiedSample Diagnosis Malignant Diagnosis 1 Benign Benign 2 Benign Benign 3Benign Benign 4 Benign Benign 5 Benign Benign 6 Benign Benign 7 BenignBenign 8 Benign Benign 9 Malignant Non-Seminoma 10 MalignantNon-Seminoma 11 Malignant Non-Seminoma 12 Malignant Non-Seminoma 13Malignant Non-Seminoma 14 Malignant Seminoma 15 Malignant Seminoma 16Malignant Seminoma 17 Malignant Seminoma

The characteristics of the transcripts are summarized as follows:

TABLE 15 Characteristics of Testicular Cancer Transcripts Transcript IDJunction Site Gene Junction 2 10744:14124  ND4L:ND5 3 7974:15496COII:Cytb 4 7992:15730 COII:Cytb 11 7775:13532 COII:ND5 12 8213:13991COII:ND5 13 9144:13816 ATPase6:ND5 15 9574:12972 COIII:ND5 1610367:12829  ND3:ND5 20 8469:13447 ATPase8:ND5 Peptidylpropyl isomeraseB (PPIB) N/A N/A

It is noted that transcripts 2, 3, 4, 7, 11, 12, 15, 16 and 20 are thesame as those discussed above with respect to Examples 3-8.

Homogenates were prepared using approximately 25 mg of frozen tissue anddiluted 1:4. The quantity of the transcripts was measured in RelativeLuminenscence Units RLU on a Glomax™ Multi Detection System (Promega).All samples were assayed in triplicate for each transcript. Backgroundmeasurements (no template) were done in triplicate as well. The analysisaccounted for background by subtracting the lower limit from the RLUvalues for the samples. Input RNA was accounted for by using the formulalog₂ a RLU−log₂ h RLU where a is the target fusion transcript and h isthe housekeeper transcript.

The analysis of the data comprised the following steps:

a) Establish CV's (coefficients of variation) for triplicate assays;acceptable if ≦15%.

b) Establish average RLU value for triplicate assays of target fusiontranscript (a) and housekeeper transcript (h).

c) Establish lower limit from triplicate value of background RLU (l).

d) Subtract lower limit (l) from (a).

e) Calculate log₂ a RLU−log₂ h RLU.

Summary of Results:

The results of the above analysis are illustrated in FIGS. 10 to 18,which comprise plots of the log₂ a RLU−log₂ h RLU against sample number.Also illustrated are the respective ROC (Receiver OperatingCharacteristic) curves determined from the results for each transcript.

While some transcripts distinguish between benign and malignanttesticular tissue, others demonstrate distinction between the tumoursubtypes of seminoma and non-seminoma and/or benign testicular tissue.It is therefore anticipated that combining transcripts from each classwill facilitate not only detection of testicular cancer but alsoclassification into subtype of seminoma or non-seminomas.

Transcript 2: There exists a statistically significant differencebetween the means (p<0.05) of the normal group and the malignantseminomas (p=0.02). Using a cutoff value of 1.5621 as demonstrated bythe ROC curve results in a sensitivity of 100% and specificity of 100%and the area under the curve is 1.00 indicating excellent test accuracy.There also exists a statistically significant difference between themeans (p<0.05) of the malignant seminomas and the malignantnon-seminomas (p=0.024). Using a cutoff value of 2.1006 as demonstratedby the ROC curve results in a sensitivity of 100% and specificity of 80%and the area under the curve is 0.90 indicating excellent test accuracy.The threshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 3: There exists a statistically significant differencebetween the means (p<0.05) of the normal group and the malignantseminomas (p=0.018). Using a cutoff value of 0.969 as demonstrated bythe ROC curve results in a sensitivity of 100% and specificity of 87.5%and the area under the curve is 0.969 indicating excellent accuracy.There also exists a statistically significant difference between themeans (p<0.05) of the malignant seminomas and the malignantnon-seminomas (p=0.017). Using a cutoff value of 1.8181 as demonstratedby the ROC curve results in a sensitivity of 100% and specificity of 80%and the area under the curve is 0.9 indicating excellent test accuracy.The threshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 4: There exists a statistically significant differencebetween the means (p<0.05) of the normal and malignant groups (p=0.034).Using a cutoff value of −9.7628 as demonstrated by the ROC curve resultsin a sensitivity of 67% and specificity of 100% and the area under thecurve is 0.833 indicating good test accuracy. The threshold value chosenmay be adjusted to increase either the specificity or sensitivity of thetest for a particular application.

Transcript 11: There exists a statistically significant differencebetween the means (p<0.05) of the normal group and the malignantseminomas (p=0.016). Using a cutoff value of 0.732 as demonstrated bythe ROC curve results in a sensitivity of 100% and specificity of 100%and the area under the curve is 1.00 indicating excellent test accuracy.There also exists a statistically significant difference between themeans (p<0.05) of the malignant seminomas and the malignantnon-seminomas (p=0.016). Using a cutoff value of 0.9884 as demonstratedby the ROC curve results in a sensitivity of 100% and specificity of 80%and the area under the curve is 0.90 indicating excellent test accuracy.The threshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 12: There exists a statistically significant differencebetween the means (p<0.1) of the normal group and the malignantseminomas (p=0.056). Using a cutoff value of 1.5361 as demonstrated bythe ROC curve results in a sensitivity of 100% and specificity of 87.5%and the area under the curve is 0.969 indicating excellent testaccuracy. There also exists a statistically significant differencebetween the means (p<0.05) of the malignant seminomas and the malignantnon-seminomas (p=0.044). Using a cutoff value of 1.6039 as demonstratedby the ROC curve results in a sensitivity of 100% and specificity of 80%and the area under the curve is 0.9 indicating excellent test accuracy.The threshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 13: There exists a statistically significant differencebetween the means (p<0.05) of the normal group and the malignant group(p=0.019). Using a cutoff value of −9.8751 as demonstrated by the ROCcurve results in a sensitivity of 87.5% and specificity of 78% and thearea under the curve is 0.875 indicating very good test accuracy. Therealso exists a statistically significant difference between the means(p<0.01) of the malignant non-seminomas and the benign group (p=0.000).Using a cutoff value of −13.9519 as demonstrated by the ROC curveresults in a sensitivity of 100% and specificity of 87.5% and the areaunder the curve is 0.975 indicating excellent test accuracy. There alsoexists a statistically significant difference between the means (p<0.01)of the malignant seminomas and the malignant non-seminomas (p=0.001).Using a cutoff value of −15.8501 as demonstrated by the ROC curveresults in a sensitivity of 100% and specificity of 100% and the areaunder the curve is 1.00 indicating excellent test accuracy. Thethreshold value chosen may be adjusted to increase either thespecificity or sensitivity of the test for a particular application.

Transcript 15: There exists a statistically significant differencebetween the means (p<0.1) of the normal and malignant groups (p=0.065).Using a cut-off value of −5.4916 as demonstrated by the ROC curveresults in a sensitivity of 75% and specificity of 89% and the areaunder the curve is 0.833 indicating good test accuracy. The thresholdvalue chosen may be adjusted to increase either the specificity orsensitivity of the test for a particular application.

Transcript 16: There exists a statistically significant differencebetween the means (p<0.05) of the normal and malignant groups includingboth seminomas and non-seminomas (p=0.037). Using a cut-off value of−6.448 as demonstrated by the ROC curve results in a sensitivity of 89%and specificity of 75% and the area under the curve is 0.806 indicatinggood test accuracy. There also exists a statistically significantdifference between the means (p<0.05) of the normal and malignantseminomas (p=0.037). Using a cut-off value of −7.4575 as demonstrated bythe ROC curve results in a sensitivity of 100% and specificity of 87.5%and the area under the curve is 0.938 indicating excellent testaccuracy. The threshold value chosen may be adjusted to increase eitherthe specificity or sensitivity of the test for a particular application.

Transcript 20: There exists a statistically significant differencebetween the means (p<0.01) of the normal group and the malignantseminomas (p=0.006). Using a cutoff value of 1.8364 as demonstrated bythe ROC curve results in a sensitivity of 100% and specificity of 100%and the area under the curve is 1.00 indicating excellent test accuracy.There also exists a statistically significant difference between themeans (p<0.01) of the malignant seminomas and the malignantnon-seminomas (p=0.004). Using a cutoff value of 1.6065 as demonstratedby the ROC curve results in a sensitivity of 100% and specificity of100% and the area under the curve is 1.00 indicating excellent testaccuracy. The threshold value chosen may be adjusted to increase eitherthe specificity or sensitivity of the test for a particular application.

Conclusions:

The above results illustrate the utility of transcripts 2, 3, 4, 11, 12,13, 15, 16, and 20 in the detection of testicular cancer, and testicularcancer subtypes, and in distinguishing malignant from normal testiculartissue. Transcript 2 was also found to have utility in the detection ofprostate, breast, colorectal and ovarian cancer. Transcript 3 was alsofound to have utility in the detection of prostate, breast, melanoma,colorectal, and ovarian cancers. Transcript 4 was also found to haveutility in the detection of prostate and colorectal cancers. Transcript11 was also found to have utility in the detection of colorectal,melanoma, and ovarian cancers. Transcript 12 was also found to haveutility in the detection of colorectal and ovarian cancers. Transcript15 was also found to have utility in the detection of melanoma andovarian cancers. Transcript 16 was also found to have utility in thedetection of melanoma skin cancer. Transcript 20 was also found to haveutility in the detection of colorectal cancer, melanoma, and ovariancancer. Any of the 9 transcripts listed may be used individually or incombination as a tool for the detection or characterization oftesticular cancer in a clinical setting.

In one aspect, the invention provides a kit for conducting an assay fordetermining the presence of cancer in a tissue sample. The kit includesthe required reagents for conducting the assay as described above. Inparticular, the kit includes one or more containers containing one ormore hybridization probes corresponding to transcripts 1 to 17, and 20described above. As will be understood, the reagents for conducting theassay may include any necessary buffers, salts, detection reagents etc.Further, the kit may include any necessary sample collection devices,containers etc. for obtaining the needed tissue samples, reagents ormaterials to prepare the tissue samples for example by homogenization ornucleic acid extraction, and for conducting the subject assay or assays.The kit may also include control tissues or samples to establish orvalidate acceptable values for diseased or non-diseased tissues.

Although the invention has been described with reference to certainspecific embodiments, various modifications thereof will be apparent tothose skilled in the art without departing from the spirit and scope ofthe invention as outlined in the claims appended hereto. All documents(articles, manuals, patent applications etc) referred to in the presentapplication are incorporated herein in their entirety by reference.

Bibliography

The following references, amongst others, were cited in the foregoingdescription. The entire contents of these references are incorporatedherein by way of reference thereto.

Author Journal Title Volume Date Anderson et al Nature Sequence andOrganization of the Human 290(5806): 457- 1981 Mitochondrial Genome 65Andrews et al Nat Genet Reanalysis and revision of the Cambridge 23(2):147 1999 reference sequence for human mitochondrial DNA. Modica- ExpertRev Mitochondria as targets for detection and 4: 1-19 2002 Napolitano etal Mol Med treatment of cancer Sherratt et al Clin Sci (Lond)Mitochondrial DNA defects: a widening 92(3): 225-35 1997 clinicalspectrum of disorders. Croteau et al Mutat Res Mitochondrial DNA repairpathways. 434(3): 137-48 1999 Green and J Clin Invest Pharmacologicalmanipulation of cell death: 115(10): 2610- 2005 Kroemer clinicalapplications in sight? 2617 Dai et al Acta Correlation of cochlear bloodsupply with 24(2): 130-6 2004 Otolaryngol mitochondrial DNA commondeletion in presbyacusis. Ro et al Muscle Nerve Deleted 4977-bpmitochondrial DNA 28(6): 737-43 2003 mutation is associated withsporadic amyotrophic lateral sclerosis: a hospital- based case-controlstudy. Barron et al Invest Mitochondrial abnormalities in ageing 42(12):3016-22 2001 Ophthalmol macular photoreceptors. Vis Sci Lewis et al JPathol Detection of damage to the mitochondrial 191(3): 274-81 2000genome in the oncocytic cells of Warthin's tumour. Muller-Hocker ModPathol The common 4977 base pair deletion of 11(3): 295-301. 1998 et almitochondrial DNA preferentially accumulates in the cardiac conductionsystem of patients with Kearns-Sayre syndrome. Porteous et al Eur JBiochem Bioenergetic consequences of accumulating 257(1): 192-201 1998the common 4977-bp mitochondrial DNA deletion. Parr et al J Mol DiagnSomatic mitochondrial DNA mutations in 8(3): 312-9. 2006 prostate cancerand normal appearing adjacent glands in comparison to age- matchedprostate samples without malignant histology. Maki et al Am J ClinMitochondrial genome deletion aids in the 129(1): 57-66 2008 Patholidentification of false- and true-negative prostate needle core biopsyspecimens. Nakase et al Am J Hum Transcription and translation ofdeleted 46(3): 418-27. 1990 Genet mitochondrial genomes in Kearns-Sayresyndrome: implications for pathogenesis. Libura et al BloodTherapy-related acute myeloid leukemia- 105(5): 2124-31 2005 like MLLrearrangements are induced by etoposide in primary human CD34+ cells andremain stable after clonal expansion. Meyer et al Proc Natl Diagnostictool for the identification of MLL 102(2): 449-54 2005 Acad Scirearrangements including unknown partner USA genes. Eguchi et al GenesMLL chimeric protein activation renders 45(8): 754-60 2006 Chromosomescells vulnerable to chromosomal damage: Cancer an explanation for thevery short latency of infant leukemia. Hayashi et al Proc NatlIntroduction of disease-related 88: 10614- 1991 Acad Sci mitochondrialDNA deletions into HeLa cells 10618 USA lacking mitochondrial DNAresults in mitochondrial dysfunction

TABLE 1 Known mitochondrial deletions having an ORF Deletion DeletionRepeat Number of Junction (nt:nt) Size (bp) Location (nt/nt) RepeatsReferences COX I - ND5 6075:13799 −7723 6076-6084/13799- D, 9/9 Mita,S., Rizzuto, R., Moraes, C. T., Shanske, S., Arnaudo, E., Fabrizi, 13807G. M., Koga, Y., DiMauro, S., Schon, E. A. (1990) “Recombination viaflanking direct repeats is a major cause of large-scale deletions ofhuman mitochondrial DNA” Nucleic Acids Research 18(3): 561-5676238:14103 −7864 6235-6238/14099- D, 4/4 Blok, R. B., Thorburn, D.R.,Thompson, G. N., Dahl, H. H. (1995) “A 14102 topoisomerase II cleavagesite is associated with a novel mitochondrial DNA deletion” HumanGenetics 95 (1): 75-81 6325:13989 −7663 6326-6341/13889- D, 16/17Larsson, N. G., Holme, E., Kristiansson, B., Oldfors, A., Tulinius, M.14004 (1990) “Progressive increase of the mutated mitochondrial DNAfraction in Kearns-Sayre syndrome” Pediatric Research 28 (2): 131- 136Larsson, N. G., Holme, E. (1992) “Multiple short direct repeatsassociated with single mtDNA deletions ” Biochimica et Biophysica Acta1139(4): 311-314 6330:13994 −7663 6331-6341/13994- D, 11/11 Mita, S.,Rizzuto, R., Moraes, C. T., Shanske, S., Arnaudo, E., Fabrizi, 14004 G.M., Koga, Y., DiMauro, S., Schon, E.A. (1990) “Recombination viaflanking direct repeats is a major cause of large-scale deletions ofhuman mitochondrial DNA” Nucleic Acids Research 18(3): 561-567 COX II -ND5 7829:14135 −6305 7824-7829/14129- D, 6/6 Bet, L, Moggio, M., Comi,G. P., Mariani, C., Prelle, A., Checcarelli, N., 14134 Bordoni, A.,Bresolin, N., Scarpini, E., Scarlato, G. (1994) “Multiple sclerosis andmitochondrial myopathy: an unusual combination of diseases” Journal ofNeurology 241 (8): 511-516 8213:13991 −5777 8214-8220/13991- D, 7/7Hinokio, Y., Suzuki, S., Komatu, K., Ohtomo, M., Onoda, M., 13997Matsumoto, M., Hirai, S., Sato, Y., Akai, H., Abe, K., Toyota, T. (1995)“A new mitochondrial DNA deletion associated with diabetic amyotrophy,diabetic myoatrophy and diabetic fatty liver” Muscle and Nerve 3 (9):S142-149 ATPase - ND5 8631:13513 −4881 8625-8631/13506- D, 7/7 Zhang,C., Baumer, A., Mackay, I. R., Linnane, A. W., Nagley, P. (1995) 13512“Unusual pattern of mitochondrial DNA deletions in skeletal muscle of anadult human with chronic fatigue syndrome” Human Molecular Genetics 4(4): 751 -754 9144:13816 −4671 9137-9144/13808- D, 8/8 Ota, Y., Tanaka,M., Sato, W., Ohno, K., Yamamoto, T., Maehara, M., 13815 Negoro, T.,Watanabe, K., Awaya, S., Ozawa, T. (1991) “Detection of plateletmitochondrial DNA deletions in Kearns-Sayre syndrome” InvestigativeOphthalmology and Visual Science 32 (10): 2667-2675 9191:12909 −37179189-9191/12906- D, 3/3 Tanaka, M., Sato, W., Ohno, K., Yamamoto, T.,Ozawa, T. (1989) 12908 “Direct sequencing of mitochondrial DNA inmyopathic patients” Biochemical and Biophysical Research Communications164 ( ): 156- 163 COX III - ND5 10190:13753  −3562 10191-10190/13753- D,8/8 Rotig, A., Bourgeron, T., Chretien. D., Rustin, P., Munnich, A.(1995) 13760 “Spectrum of mitochondrial DNA rearrangements in thePearson marrow-pancreas syndrome” Human Molecular Genetics 4 (8): 1327-1330 Rotig, A., Cormier, Y., Kol, F., Mize, C. E., Souslubray, J. M.,Veerman, A., Pearson, H. A., Munnich, A. (1991) “Site-specific deletionsof the mitochondrial genome in Pearson marrow-pancreas syndrome”Genomics 10 (2): 502-504 10067:12029  −2461 10365-10367/12825- D, 3/3Kapsa, R., Thompson, G. N., Thorburn, D. R., Dahl, H. H., Marzuki, G.,12828 Byrna, E., Blair, R. B. (1984) “A novel mtDNA deletion in aninfant with Pearson syndrome” Journal of Inherited Metabolic Disease 17(5): 521- 526 ND4L - ND5 10744:14124  −3378 10745-10754/14124- D, 9/10Cormier-Daire, V., Bonnefont, J. P., Rustin, P., Maurage, C., Ogler, H.,14133 Schmitz, J., Ricour, C., Saudubray, J. M., Munnich, A., Rotig, A.(1984) “Mitochondrial DNA, rearrangements with onset as chronic diarrheawith villous atrophy” Journal of Pediatrics 124 (1): 53-70 ND4 - ND511232:13980  −2747 1324-11242/13981- D, 9/9 Rotig, A., Cormier, V.,Roll, F., Mize, C. E., Saudubray, J. M., Veerman, 13989 A., Pearson, H.A., Munnich, A. (1991) “Site-specific deletions of the mitochondrialgenome in Pearson marrow-pancreas syndrome” Genomics 10 (2): 502-504Rotig, A., Cormier, Y., Blanche, S., Bonnefont, J. P., Ledeist, F.,Romero, N., Schmitz, J., Rustin, P., Fischer, A., Saudubray, J. M.(1990) “Pearson's marrow-pancreas syndrome. A multi-system mitochondrialdisorder in infancy” Journal of Clinical Investigation 86 ( ): 1601-1608Cormier, V., Rotig, A., Quartino, A. R., Forni, G. L., Cerane, R.,Maier, M., Saudubray, J. M., Munnich, A. (1990) “Widespread multitissuedeletions of the mitochondrial genome in Pearson marrow-pancreassyndrome” Journal of Pediatrics 117 (4): 599-602 Awata, T., Matsumata,T., Iwamoto, Y., Matsuda, A., Kuzuya, T., Saito, T. (1993) “Japanesecase of diabetes mellitus and deafness with mutations in mitochondrialtRNALeu(UUR) gene [letter]” Lancet 341 (8855): 1281-1282

TABLE 2 Prostate Cancer Detection with Novel Mitochondrial FusionTranscripts

* unit results in table are RLU (relative luminescence units); Data readon Glorunner ™. % CV = Coefficient of variation (as %). Legend: Homog =homogenate. Homog 1: Prostate tumour tissue sample from patient; Homog2: Histologically normal tissue adjacent to tumour from patient. RNA:Control: Total RNA from prostate tissue (Ambion p/n 7988). Shading:Background measurement.

TABLE 3 Deletion/Transcript/DNA Complement DNA sequence with deletioncomplementary Deletion RNA transcript to RNA transcript Transcript No.ATP synthase F0 subunit 8 to NADH SEQ ID NO: 18 SEQ ID NO: 2 1dehydrogenase subunit mitochondrial positions 8366-14148 (with referenceto SEQ ID NO: 1). NADH dehydrogenase subunit 4L SEQ ID NO: 19 SEQ ID NO:3 2 (ND4L) to NADH dehydrogenase subunit 5 (ND5); Mitochondrialpositions 10470- 14148 (with reference to SEQ ID NO: 1) Cytochrome coxidase subunit II (COII) to SEQ ID NO: 20 SEQ ID NO: 4 3 Cytochrome b(Cytb); Mitochondrial positions 7586-15887 (with reference to SEQ IDNO: 1) Cytochrome c oxidase subunit II (COII) to SEQ ID NO: 21 SEQ IDNO: 5 4 Cytochrome b (Cytb); Mitochondrial positions 7586-15887 (withreference to SEQ ID NO: 1)

TABLE 4 Breast and Prostate Cancer Detection Normal Normal Normaladjacent Adjacent Adjacent Breast Breast Breast to Breast ProstateProstate Prostate to Prostate Tumour 1 Tumour 1 Tumour 2 Tumour 2 Tumour3 Tumour 4 Tumour 5 Tumour 5 1 2 3 4 5 6 7 8 1:100 dilution E 68920 297149108 1245 46723 56679 99836 35504 1:100 dilution replicate F 92409 301760637 1512 53940 56155 100582 44221 G 420 3 31 6 26 25 44 23 H 518 3 4 55 3 4 2 % CV 20.6 1.1 14.9 13.7 10.1 0.7 0.5 15.5 unit results in tableare RLU (relative luminescence units) background G1, H1 empty wellG2-G8, H2-H8

TABLE 5a Assay Conditions Template for the assay Homo- Homo- Homo- Homo-Homo- Homo- Homo- Homo- RNA gen 1 gen 2 RNA gen 1 gen 2 RNA gen 1 gen 2RNA gen 1 gen 2 Tran- Tran- Tran- Tran- Tran- Tran- Tran- Tran- Tran-Tran- Tran- Tran- script 1 script 1 script 1 script 2 script 2 script 2script 3 script 3 script 3 script 4 script 4 script 4 1 2 3 4 5 6 7 8 910 11 12 A RNA Homog 1 Homog 2 RNA Homog 1 Homog 2 RNA Homog 1 Homog 2RNA Homog 1 Homog 2 B Dil 1 Dil 1 Dil 1 Dil 1 Dil 1 Dil 1 Dil 1 Dil 1Dil 1 Dil 1 Dil 1 Dil 1 C RNA Homog 1 Homog 2 RNA Homog 1 Homog 2 RNAHomog 1 Homog 2 RNA Homog 1 Homog 2 D Dil 2 Dil 2 Dil 2 Dil 2 Dil 2 Dil2 Dil 2 Dil 2 Dil 2 Dil 2 Dil 2 Dil 2 E RNA Homog 1 Homog 2 RNA Homog 1Homog 2 RNA Homog 1 Homog 2 RNA Homog 1 Homog 2 F Dil 3 Dil 3 Dil 3 Dil3 Dil 3 Dil 3 Dil 3 Dil 3 Dil 3 Dil 3 Dil 3 Dil 3 G RNA Homog 1 Tran-RNA Homog 1 Tran- RNA Homog 1 Tran- RNA Homog 1 Tran- script 1 script 1script 1 script 1 H Dil 4 Dil 4 Back- Dil 4 Dil 4 Back- Dil 4 Dil 4Back- Dil 4 Dil 4 Back- ground ground ground ground Homogenate1- Used 26mg of tissue to homogenize in 700 ul H soln with Proteinase K (PK). UsedQiagen TissueRuptor. Used 40 ul homogenate supernatant, 20, 10 and 5 ulfor dilution Homogenate1 = Tumour tissue from the tumorous ProstateHomogenate2- Used 29 mg of tissue to homogenize in 700 ul H soln withPK. Used Qiagen TissueRuptor. Used 40 ul homogenate supernatant, 20, 10and 5 ul for dilution Homogenate2= Normal tissue from the tumorousProstate RNA dilution was made as below. RNA was from Prostate Normalfrom Ambion. Assay was done in duplicates.

TABLE 5b RNA dilution RNA Dilution ng/ul Dil 1 3000 1:3 dil Dil 2 1000Serial dil Dil 3 333 Dil 4 111

TABLE 6 Transcript Summary by Disease Prostate Breast ColorectalMelanoma Lung Ovarian Testicular Probe Cancer Cancer Cancer Skin CancerCancer Cancer Cancer 1 • • 2 • • • • • 3 • • • • 4 • • 5 6 • • • 7 8 • •9 • 10 • • • 11 • • • • 12 • • • 13 • 14 • 15 • • • 16 • • 17 20 • • • •

1. An isolated mitochondrial fusion transcript associated with cancer.2. The mitochondrial fusion transcript of claim 1, wherein thetranscript comprises an insertion, translocation, deletion, duplication,recombination, rearrangement or combination thereof.
 3. Themitochondrial fusion transcript of claim 2, wherein the transcriptcomprises a deletion.
 4. The mitochondrial fusion transcript of claim 3,wherein the transcript comprises a sequence as set forth in any one ofSEQ ID NOs:18 to 33 or
 50. 5. The mitochondrial fusion transcript ofclaim 3, wherein the transcript comprises a sequence as set forth in anyone of SEQ ID NOs: 18-21, 23, 25-33 or
 50. 6. The mitochondrial fusiontranscript of claim 3, wherein the transcript comprises the expressedRNA transcript of a deletion sequence set out in Table
 1. 7. Amitochondrial fusion protein corresponding to the fusion transcript ofclaim 4 and having a sequence as set forth in any one of SEQ ID NOs: 34to 49 and
 52. 8. An isolated mitochondrial DNA (mtDNA) encoding thefusion transcript of claim
 1. 9. The isolated mtDNA of claim 8 having asequence as set forth in any one of SEQ ID NOs: 2-17 or
 51. 10. Ahybridization probe having a nucleic acid sequence complementary to atleast a portion of the mitochondrial fusion transcript according toclaim
 4. 11. A method of detecting a cancer in a mammal, the methodcomprising assaying a tissue sample from the mammal for the presence ofat least one mitochondrial fusion transcript associated with cancer byhybridizing the sample with at least one hybridization probe having anucleic acid sequence complementary to at least a portion of themitochondrial fusion transcript according to claim
 4. 12. A method ofdetecting a cancer in a mammal, the method comprising assaying a tissuesample from the mammal for the presence of at least one aberrant mtDNAassociated with cancer by hybridizing the sample with at least onehybridization probe having a nucleic acid sequence complementary to atleast a portion of the mtDNA according to claim
 7. 13. The method ofclaim 11, wherein the cancer is prostate cancer, testicular cancer,ovarian cancer, breast cancer, colorectal cancer, lung cancer, melanomaskin cancer or combinations thereof.
 14. The method of claim 13, whereinthe assay comprises: a) conducting a hybridization reaction using atleast one of said probes to allow said at least one probe to hybridizeto a complementary mitochondrial fusion transcript or mtDNA; b)quantifying the amount of the at least one mitochondrial fusiontranscript or mtDNA in said sample by quantifying the amount of saidtranscript or mtDNA hybridized to said at least one probe; and, c)comparing the amount of the mitochondrial fusion transcript or mtDNA inthe sample to at least one known reference value.
 15. The method ofclaim 14, wherein the assay is carried out using diagnostic imagingtechnology.
 16. The method of claim 15, wherein the diagnostic imagingtechnology comprises high throughput microarray analysis.
 17. The methodof claim 14, wherein the assay is carried out using branched DNAtechnology.
 18. The method of claim 14, wherein the assay is carried outusing PCR.
 19. A kit for conducting an assay for detecting the presenceof a cancer in a mammal, said kit comprising at least one hybridizationprobe complementary to at least a portion of the fusion transcript ofclaim
 4. 20. A screening tool comprised of a microarray having 10's,100's, or 1000's of mitochondrial fusion transcripts according to claim4 for identification of those associated with cancer.
 21. A screeningtool comprised of a microarray having 10's, 100's, or 1000's ofmitochondrial DNAs according to claim 9 for identification of thoseassociated with cancer.
 22. A screening tool comprised of a multiplexedbranched DNA assay having 10's, 100's, or 1000's of mitochondrial fusiontranscripts according to claim 4 for identification of those associatedwith cancer.
 23. A screening tool comprised of a multiplexed branchedDNA assay having 10's, 100's, or 1000's of mitochondrial DNAs accordingto claim 9 for identification of those associated with cancer.
 24. Ahybridization probe having a nucleic acid sequence complementary to atleast a portion of the mitochondrial fusion transcript according toclaim
 5. 25. A hybridization probe having a nucleic acid sequencecomplementary to at least a portion of a mitochondrial fusion transcriptaccording to claim 3, wherein the transcript comprises the expressed RNAtranscript of a deletion sequence set out in Table
 1. 26. Ahybridization probe having a nucleic acid sequence complementary to atleast a portion of the mtDNA of claim
 9. 27. A method of detecting acancer in a mammal, the method comprising assaying a tissue sample fromthe mammal for the presence of at least one mitochondrial fusiontranscript associated with cancer by hybridizing the sample with atleast one hybridization probe having a nucleic acid sequencecomplementary to at least a portion of the mitochondrial fusiontranscript according to claim
 5. 28. A method of detecting a cancer in amammal, the method comprising assaying a tissue sample from the mammalfor the presence of at least one mitochondrial fusion transcriptassociated with cancer by hybridizing the sample with at least onehybridization probe having a nucleic acid sequence complementary to atleast a portion of the mitochondrial fusion transcript according to 3,wherein the transcript comprises the expressed RNA transcript of adeletion sequence set out in Table
 1. 29. A method of detecting a cancerin a mammal, the method comprising assaying a tissue sample from themammal for the presence of at least one aberrant mtDNA associated withcancer by hybridizing the sample with at least one hybridization probehaving a nucleic acid sequence complementary to at least a portion ofthe mtDNA according to claim
 9. 30. The method of claim 27, wherein thecancer is prostate cancer, testicular cancer, ovarian cancer, breastcancer, colorectal cancer, lung cancer, melanoma skin cancer orcombinations thereof.
 31. The method of claim 28, wherein the cancer isprostate cancer, testicular cancer, ovarian cancer, breast cancer,colorectal cancer, lung cancer, melanoma skin cancer or combinationsthereof.
 32. The method of claim 12, wherein the cancer is prostatecancer, testicular cancer, ovarian cancer, breast cancer, colorectalcancer, lung cancer, melanoma skin cancer or combinations thereof. 33.The method of claim 29, wherein the cancer is prostate cancer,testicular cancer, ovarian cancer, breast cancer, colorectal cancer,lung cancer, melanoma skin cancer or combinations thereof.
 34. A kit forconducting an assay for detecting the presence of a cancer in a mammal,said kit comprising at least one hybridization probe complementary to atleast a portion of the fusion transcript of claim
 5. 35. A kit forconducting an assay for detecting the presence of a cancer in a mammal,said kit comprising at least one hybridization probe complementary to atleast a portion of the fusion transcript of claim 3, wherein thetranscript comprises the expressed RNA transcript of a deletion sequenceset out in Table
 1. 36. A kit for conducting an assay for detecting thepresence of a cancer in a mammal, said kit comprising at least onehybridization probe complementary to at least a portion of the mtDNA ofclaim
 9. 37. A screening tool comprised of a microarray having 10's,100's, or 1000's of mitochondrial fusion transcripts according to claim5 for identification of those associated with cancer.
 38. A screeningtool comprised of a microarray having 10's, 100's, or 1000's ofmitochondrial fusion transcripts according to claim 3 for identificationof those associated with cancer, wherein the transcript comprises theexpressed RNA transcript of a deletion sequence set out in Table
 1. 39.A screening tool comprised of a multiplexed branched DNA assay having10's, 100's, or 1000's of mitochondrial fusion transcripts according toclaim 5 for identification of those associated with cancer.
 40. Ascreening tool comprised of a multiplexed branched DNA assay having10's, 100's, or 1000's of mitochondrial fusion transcripts according toclaim 3 for identification of those associated with cancer, wherein thetranscript comprises the expressed RNA transcript of a deletion sequenceset out in Table 1.